Lycopersicon assays of chemical/radiation genotoxicity for the study of environmental mutagens

From a literature survey, 21 chemicals are tabulated that have been evaluated in 39 assays for their clastogenic effects in Lycopersicon. Nineteen of the 21 chemicals are reported as giving a positive reaction (i.e. causing chromosome aberrations). Of these, five are reported positive with a dose response. In addition, 23 assays have been recorded for six types of radiation, all of which reacted positively. The results of 102 assays with 32 chemicals and seven types of radiation tested for the induction of gene mutations are tabulated, as well as 20 chemicals and/or radiation in combined treatments. The Lycopersicon esculentum (2n=24) assay is a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis and for somatic mutations induced by chemicals and radiations. The Lycopersicon bioassay has been shown to be as sensitive and as specific an assay as other plant genotoxicity assays, such as Hordeum vulgare, Vicia faba, Crepis capillaris, Pisum sativum and Allium cepa and should be considered in further studies in assessing clastogenicity. Tests using L. esculentum can be made for a spectrum of mutant phenotypes of which many are identifiable in young seedlings.     

Chromosome aberration assays in Pisum for the study of environmental mutagens

From a literature survey, 117 chemicals are tabulated that have been assayed in 179 assays for their clastogenic effects in Pisum. Of the 117 chemicals that have been assayed, 65 are reported at giving a positive reaction (i.e. causing chromosome aberrations), 30 positive with a dose response, five borderline positive. Seventeen chemicals gave a negative response. Eighty-one percent of the chemicals gave a definite positive response. A c-mitotic effect was detected from treatment with 17 chemicals. In addition to the above tabulation of chemicals, 39 chemicals have been reported with an antimitotic effect. Thirteen assays have been recorded for five types of radiation, which with the exception of ultrasound reacted positively. The results of assays with 38 chemicals and/or radiations in combined treatments, as well as 15 chemicals and three types of radiations that induce somatic mutations are tabulated. The Pisum sativum (2n=14) bioassay has been shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis for somatic mutations induced by chemicals, radiations, and environmental pollutants. For some chemicals, the Pisum assay is not as sensitive in assessing clastogenicity as the Allium assay, although this should be considered in relative terms. Pisum fulvum (2n=14) has been used in clastogenic studies also, but to a much lesser extent.     

An evaluation of the genotoxic properties of herbicides following plant and animal activation

Commercial and technical grades of 11 herbicides and 13 combinations of commercial grade herbicides were evaluated for their genotoxic properties with Salmonella typhimurium, Saccharomyces cerevisiae directly and following plant and animal activation, or with Zea mays. The herbicides were related by their use in commercial corn (maize) production. Commercial grade formulations of each herbicide and combination of herbicides were also evaluated in situ with the pollen waxy locus assay of Z. mays. Eradicane and bifenox were negative in all assays. Alachlor, propachlor, procyazine and SD50093 (a formulation of cyanazine plus atrazine) were positive in one assay. Cyanazine, dicamba and metolachlor were positive in 2 assays. Atrazine, simazine and butylate were tested only in situ. Atrazine and simazine were positive and butylate was negative. Of the combinations of herbicides evaluated with the 3 genetic assays, alachlor plus bifenox and procyazine plus metolachlor were positive in 1 assay and metolachlor plus atrazine was positive in 2 assays. Of the combinations of herbicides evaluated only in situ, butylate plus atrazine, eradicane plus atrazine, eradicane plus cyanazine and metolachlor plus cyanazine were positive while butylate plus cyanazine was negative.     

The present status of higher plant bioassays for the detection of environmental mutagens

Higher plants provide valuable genetic assay systems for screening and monitoring environmental pollutants. They are now recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and are applicable for the detection of environmental mutagens both indoor and outdoor. Comparisons between plant and nonplant genetic assay systems indicate that higher plant genetic assays have a high sensitivity (i.e. few false negatives). Two assays which are considered ideal for in situ monitoring and testing of airborne and aqueous mutagenic agents are the Tradescantia stamen hair assay for mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing. Other higher plant gentoxicity assys which have a large number of genetic markers and/or data base and are also highly suitable for testing for genotoxic agents include Arabidopsis thaliana, Allium cepa, Hordeum vulgare, Vicia faba, and Zea mays. Since higher plant systems are now recognized as excellent indicators of the cytotoxic, cytogenetic, and mutagenic effects of environmental chemicals and have unique advantages for in situ monitoring and screening it is recommended that higher plant systems be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damage resulting from pollution or the use of environmental chemicals. The results from higher platn genetic assays could meke a significant contribution in protecting the public from agents that can cause mutation and cancer. The advantages possessed by higher plant genetic assays, which are inexpensive and easy to handle, make them ideal for use by scientists in developing countries.     

Production and characterization of maize chromosome 9 radiation hybrids derived from an oat-maize addition line

In maize (Zea mays L., 2n = 2x = 20), map-based cloning and genome organization studies are often complicated because of the complexity of the genome. Maize chromosome addition lines of hexaploid cultivated oat (Avena sativa L., 2n = 6x = 42), where maize chromosomes can be individually manipulated, represent unique materials for maize genome analysis. Maize chromosome addition lines are particularly suitable for the dissection of a single maize chromosome using radiation because cultivated oat is an allohexaploid in which multiple copies of the oat basic genome provide buffering to chromosomal aberrations and other mutations. Irradiation (gamma rays at 30, 40, and 50 krad) of a monosomic maize chromosome 9 addition line produced maize chromosome 9 radiation hybrids (M9RHs)-oat lines possessing different fragments of maize chromosome 9 including intergenomic translocations and modified maize addition chromosomes with internal and terminal deletions. M9RHs with 1 to 10 radiation-induced breaks per chromosome were identified. We estimated that a panel of 100 informative M9RHs (with an average of 3 breaks per chromosome) would allow mapping at the 0. 5- to 1.0-Mb level of resolution. Because mapping with maize chromosome addition lines and radiation hybrid derivatives involves assays for the presence or absence of a given marker, monomorphic markers can be quickly and efficiently mapped to a chromosome region. Radiation hybrid derivatives also represent sources of region-specific DNA for cloning of genes or DNA markers.     

An introduction and study design

Under the sponsorship of the International Programme on Chemical Safety (IPCS), 17 laboratories from diverse regions of the world participated in evaluating the utility of four plant bioassays for detecting genetic hazards of environmental chemicals. The bioassays included in this collaborative study were: Arabidopsis thaliana embryo and chlorophyll assay and Tradescantia stamen hair assay, Tradescantia paludosa micronucleus assay and Vicia Faba root tip assay. Four to six laboratories participated in the performance of each of the bioassays. All laboratories participating in a particular bioassay were supplied with uniform plant material as well as standardized protocol. Five direct acting water soluble test chemicals, i.e. maleic hydrazide, methyl nitrosourea, ethyl methanesulfonate, sodium azide and azidoglycerol, were selected for this study. The study was designed to be completed in three phases. Ethyl methanesulfonate was used as a positive control and has already been reported earlier (Sandhu et al., 1991). The data from the remaining four chemicals used for the evaluation of four plant test systems in the first phase of the collaborative study are reported in this issue.     

Combined cycloheximide and 8-hydroxyquinoline pretreatment for study of plant chromosomes.

The actions of cycloheximide and 8-hydroxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydroxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.     

Physical mapping of the 5S and 45S rDNA in teosintes

The physical locations of the 5S and 45S rDNA sequences were examined in three types of teosinte, Zea mays ssp. mexicana (2n = 20), Zea diploperennis (2n = 20) and Zea perennis (2n = 40) by biotinylated fluorescence in situ hybridization (FISH). The tested materials only showed one hybridization site of 5S rDNA on their genomes, but they were different in the position of the signals. The hybridization site of Zea mays ssp. mexicana was located on the long arm of chromosome 2, indicating that it is the same as the cultivated maize in the position of 5S rDNA, while the sites of Zea diploperennis and Zea perennis were on the short arms of other chromosomes. For 45S rDNA, one hybridization site was detected at secondary constriction region of the satellite chromosomes in Zea mays ssp. mexicana and Zea diploperennis, while in Zea perennis, besides the site located at the secondary constriction region, a second site on the short arm of another chromosome pair was observed. Our results provide additional evidence for Zea mays ssp. mexicana being a subspecies of Zea mays.     

Review of literature on chemical-induced aneuploidy in mammalian male germ cells

80 papers published between 1970 and 1984 were evaluated for results pertaining to chemical-induced aneuploidy in mammalian male germ cells. Diverse assays and end points were represented. The assays considered to involve direct measures of aneuploidy were based upon chromosome counts in premeiotic, meiotic, and embryonic cells, and the male pronucleus, or upon phenotypic expression of X-linked genetic markers. Assays in which indirect measures were interpreted as evidence for aneuploidy included those primarily assessing chiasma frequencies, univalent frequencies, and spermatid/sperm sex chromosome body counts. An initial screening to reject studies with insufficient data and those which did not involve a single chemical test agent led to the elimination of 39 papers from further review. The remaining 41 papers reported effects from 46 different chemicals. These papers were rigorously assessed for adequacy of experimental protocols, relevance of end points as direct measures of aneuploidy, and completeness of data presentation and statistical analysis. Criteria specific to each assay were also considered. 4 chemical tests were considered to provide reliable positive or negative aneuploidy data. Cyclophosphamide and chloral hydrate each caused metaphase II hyperploidy when injected into mice. Very limited analyses of trenimon and isoniazid provided negative results. Test findings for 44 chemicals were viewed as inconclusive. It was concluded that standardization of tests to evaluate chemical-induced aneuploidy in male germ cells and the application of these tests towards increasing the data base are badly needed.     

Implications for genetic toxicology of the chromosomal breakage syndromes

The activation of oncogenes and our knowledge of the chromosome breakage syndromes show that both intragenic mutations and chromosomal aberrations are important in carcinogenesis. Each suggests that an agent could produce genetic changes in a tissue without producing cancer there, if the types of genetic change do not match: chromosomal aberrations may be irrelevant in the mammary epithelium but be very significant in the bone marrow, and vice versa. This has vital implications for genetic toxicology: (1) both gene mutations and chromosomal aberrations should be measured, and (2) carcinogens may be mutagenic in tissues in which they are not carcinogenic. One might therefore expect in vivo assays for mutagenicity to correlate rather well with cancer bioassays; unfortunately, the bioassays themselves seem faulty. If cancer bioassays are valid, they would be reproducible. If bioassays are reproducible, they would be internally consistent. The information supplied by Tennant et al. (1987) for their validation of in vitro assays gives data from both sexes in rats and mice for 70 chemicals. When the data are analyzed site-by-site, positive results were not replicated in the other sex or in the other species much of the time: in half the cases the other sex does not give the same result; in two-thirds of the cases the other species does not give the same result. There are 3 potential explanations for these differing results: (1) genuine sex-specific carcinogens are common, (2) genuine species-specific carcinogens are common, or (3) the bioassay does not replicate well, i.e., is erratic. The third possibility best explains the data. The apparent inability of short-term in vitro tests to discriminate well between carcinogens and non-carcinogens may be more a reflection of the cancer bioassays that were used to determine which chemicals were carcinogenic than any defect in the assays. In this situation in vivo assays can scarcely be expected to do better even if they are better.     

多枝赖草谷胱甘肽还原酶cDNA片段的克隆与分析

从盐胁迫处理的多枝赖草(Leymus multicaulis)新鲜叶片中提取分离出RNA,然后根据报道的多种植物谷胱甘肽还原酶氨基酸序列上两个保守区设计简并引物。RT-PCR获得了1条大小约400bp的条带,回收该条带并进行TA克隆,蓝白斑筛选,得到阳性克隆。经过质粒大小比较和PCR验址．进行序列测定和分析,发现该序列属于GR基因片段,其Genbank注册号为AY781786．编码的氨基酸序列与Oryza sativa、Zea mays、Arabidopsis thaliana和Nicotiana tabacum的GR相应区段的氨基酸序列一致性分别为91％、89％、86％和83％。蛋白质序列分析发现该序列含有一个吡啶二硫酸核苷酸氧化还原酶(pyridine nucleotide-disulphide oxideoreductase)保守结构域。进化树分析表明,该多枝赖草cDNA片段编码的氨基酸序列在进化上与水稻和玉米较近。     

Antimutagenic activity of Armoracia rusticana, Zea mays and Ficus carica plant extracts and their mixture

Antimutagenic action of plant extracts of Armoracia rusticana, Ficus carica, Zea mays and their mixture on environmental xenobiotics has been investigated. The plant extracts and their mixture decreased the level of mutations induced by N-metil-N'-nitro-N-nitrozoguanidin (MNNG) in Vicia faba cells, chlorophyll mutations in Arabidopsis thaliana and NaF induced mutability in rat marrow cells. The studied plant extracts and their mixture demonstrate the ability to decrease the genotoxicity of environmental mutagens.     

Antimutagenic activity of plant extracts from Armoracia rusticana, Ficus carica and Zea mays and peroxidase in eukaryotic cells

Antimutagene activity and high efficiency of antimutagene action of plant extracts from horseradish roots (Armoracia rusticana), fig brunches (Ficus carica) and mays seedlings (Zea mays) and their ability to decrease the frequency of spontaneous and induced by gamma-rays chromosome aberrations in meristematic cells of Vicia faba and marrow cells of mice have been shown. Comparative assessment of genoprotective properties of peroxidase and the studied extracts has revealed higher efficiency of antimutagene action of peroxidase.     

玉米化感物质异羟肟酸的研究进展

介绍了异羟肟酸在玉米植株中的分布和玉米根系分泌物中异羟肟酸的分析方法．丁布(DIM-BOA)是玉米植株中含量最大的异羟肟酸．不同玉米品种之间异羟肟酸含量的差异很大．种子不含异羟肟酸;但萌发后其含量迅速增加,在萌芽几天后的幼苗植株其含量达最大值,随后逐渐下降;在玉米生长发育的不同时期,幼嫩叶片内异羟肟酸含量始终较高;地上部分异羟肟酸的浓度高于根系．植株异羟肟酸的浓度受生长环境条件影响显著,在紫外辐射、黑暗条件或水分胁迫下其含量明显增加．在各种禾谷类作物中,玉米根系分泌物内含异羟肟酸较高;铁的存在能显著增加玉米根系分泌物中异羟肟酸的含量．     

GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests

A recent ECVAM workshop considered how to reduce falsely predictive positive results when undertaking in vitro genotoxicity testing, and thus to avoid unnecessary follow-up with tests involving animals. As it was anticipated that modified versions of existing assays as well as new assays might contribute to a solution, an expert panel was asked to identify a list of chemicals that could be used in the evaluation of such assays. Three categories of test chemicals were chosen comprising a total of 62 compounds. This paper provides test results for these chemicals using the GreenScreen HC assay. All tests were carried out in triplicate, by multiple operators, with and without S9, using invariant protocols. Group 1 chemicals should be detected as positive in in vitro mammalian cell genotoxicity tests: 18/20 (90%) were reproducibly positive in GreenScreen HC. Group 2 chemicals should give negative results in in vitro genotoxicity tests: 22/23 (96%) were reproducibly negative in GreenScreen HC. Overall concordance for Groups 1 and 2 is 93%. Group 3 chemicals should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce chromosomal aberrations or Tk mutations in mouse lymphoma cells, often at high concentrations or at high levels of cytotoxicity: 13/17 (76%) were reproducibly negative in GreenScreen HC. Of the four positive compounds in Group 3, p-nitrophenol was only positive at the top dose (10 mM), 2,4-DCP is an in vivo genotoxin, and two chemicals are antioxidant compounds that may be acting as pro-oxidants in the hyperoxic conditions of cell culture. Overall, these predictive figures are similar to those from other studies with the GreenScreen HC assay and confirm its high specificity, which in turn minimizes the generation of falsely predictive positive results.     

Rate and pattern of mutation at microsatellite loci in maize

Microsatellites are important tools for plant breeding, genetics, and evolution, but few studies have analyzed their mutation pattern in plants. In this study, we estimated the mutation rate for 142 microsatellite loci in maize (Zea mays subsp. mays) in two different experiments of mutation accumulation. The mutation rate per generation was estimated to be 7.7 x 10(-4) for microsatellites with dinucleotide repeat motifs, with a 95% confidence interval from 5.2 x 10(-4) to 1.1 x 10(-3). For microsatellites with repeat motifs of more than 2 bp in length, no mutations were detected; so we could only estimate the upper 95% confidence limit of 5.1 x 10(-5) for the mutation rate. For dinucleotide repeat microsatellites, we also determined that the variance of change in the number of repeats (sigma(m)2) is 3.2. We sequenced 55 of the 73 observed mutations, and all mutations proved to be changes in the number of repeats in the microsatellite or in mononucleotide tracts flanking the microsatellite. There is a higher probability to mutate to an allele of larger size. There is heterogeneity in the mutation rate among dinucleotide microsatellites and a positive correlation between the number of repeats in the progenitor allele and the mutation rate. The microsatellite-based estimate of the effective population size of maize is more than an order of magnitude less than previously reported values based on nucleotide sequence variation.     

Induced cytomictic diversity in maize (Zea mays L.) inbred

Mutation breeding has been used for improving oligogenic and polygenic characters, disease resistance and quantitative characters including yielding ability. The cytological stability of maize inbred lines is an important consideration in view of their extensive use in genetics and plant breeding research. Investigation in Zea mays L. confirms that the migration of chromosomes is a real event that cannot be misunderstood as an artifact produced by fixation or mechanical injuries. During present investigation, we found that out of six inbred lines of Zea mays L. viz. CM-135, CM-136, CM-137, CM-138, CM-142 and CM-213 at various treatment doses of gamma irradiations viz. 200, 400 and 600 Gy, some of the plants of inbred line CM- 138 at 200 Gy dose displayed characteristic cytoplasmic connections during all the stages of meiosis. Four plants from this treatment set were found to be engaged in a rare phenomenon reported as "Cytomixis". It elucidates that in inbred of Zea mays L., induced cytomixis through gamma rays treatment may be considered to be a possible source of production of aneuploid and polyploid gametes. This phenomenon may have several applications in Zea mays L. improvement in the sense of diversity and ever yield potential.     

Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage.

Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage.     

Chromosome C-banding of the teosinte Zea nicaraguensis and comparison to other Zea species

The Nicaraguan teosinte Zea nicaraguensis was studied cytologically to determine its chromosome number and C-banding pattern. The C-banding pattern was compared with that of the close relative Zea luxurians as well as with Zea diploperennis and cultivated maize, Zea mays ssp. mays. Karyograms were constructed for the four Zea species. It is shown that Z. nicaraguensis, like most other Zea species, is a diploid with 2n=20 chromosomes. The C-banding pattern shows that Z. nicaraguensis is very similar to Z. luxurians and more similar to Z. luxurians than to Z. diploperennis and cultivated maize. Whether or not Z. nicaraguensis and Z. luxurians should be regarded as subspecies instead of individual species is, however, not possible to conclude from this study.     

A report of the U.S. Environmental Protection Agency Gene-Tox Program. Evaluation of mutagenicity assays for purposes of genetic risk assessment

For the vast majority of chemicals, mammalian germ-line (MG) mutation data do not exist. The question was examined of how best to utilize results of non-MG genotoxicity assays that are included in the Gene-Tox data base to provide information of the likelihood that genetic damage might be induced in and transmitted by the reproductive cells of exposed human beings. Two approaches were used to assess the relative value of different assays for genetic hazard identification. (1) Test results were weighted according to parameters by which conditions of an assay resemble those encountered in the potential induction of transmitted genetic damage in mammals. For this purpose, 35 assays were grouped into 16 categories that were assigned weights ranging from 1 to 15; there were 2367 chemicals in the data base. This system was evaluated by comparing the sum of weighted test results for each chemical with the outcome of MG-standard (MGst) tests where such had been reported. (MGst tests used were the specific-locus and heritable-translocation assays [SLT and HTT] for gene mutations and chromosome aberrations, respectively.) The weighting system produced a few false positives with respect to the MGst results. It produced no false negatives, but the available evidence is limited by the circumstance that MGst test have evidently been preferentially performed with chemicals that had already been shown to be positive in several other assays. (2) Findings from each MGst test were compared with those from each of the other assays in turn, provided that at least 10 chemicals had been tested in both of the assays. There were 11 such comparisons involving the SLT, and 14 such comparisons involving the HTT. The observed concordance was above random expectation in several comparisons, particularly those involving certain mammalian in vivo tests, but in only one case (HTT vs. unscheduled DNA synthesis in the testis) did the degree of elevation approach statistical significance.