Shahbeg S. Sandhu

Tradescantia micronucleus bioassay.

Four coded chemicals, azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN3), and maleic hydrazide (MH), were tested with the Tradescantia micronucleus (Trad-MCN) bioassay by five independent laboratories from five different countries. The purpose of this international collaborative study was to evaluate four plant bioassays, of which the Trad-MCN assay was one, for their sensitivity, efficiency and reliability. The study was carried out under the sponsorship of the International Programme on Chemical Safety. All laboratories adhered to a standard Trad-MCN protocol which suggested that three replicate tests be conducted with each chemical. The results reported by all laboratories, although not equal, showed good agreement among the laboratories. In fact, all five laboratories obtained positive results with MH and MNU, while four of the five laboratories achieved positive results with NaN3. AG was tested in only three laboratories. Two reported negative results, while one reported positive results but only at a single high dose. The data from this study suggest that under normal conditions, the Trad-MCN bioassay is an efficient and reliable short-term bioassay for clastogens. It is suitable for the rapid screening of chemicals, and also is specially qualified for in situ monitoring of ambient pollutants.

Study of Pesticide Genotoxicity

With a limited supply of arable land supporting an ever-increasing human population, the threat of crop loss to agricultural pests becomes continually more acute. Thus pesticides have become an essential component of modern agriculture. As competing organisms evolve resistance to commonly used agents, new and more effective poisons and repellants must constantly be developed. The fundamental problem in pesticide development is to produce chemicals that act specifically against certain organisms without adversely affecting others. Because of the similarities in the structural, metabolic and genetic components of all life forms, absolute species specificity is frequently difficult to attain. Furthermore, such toxic chemicals improperly used may engender biological effects beyond those for which they were originally manufactured.

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: An assay for the detection of aneuploidy-inducing agents

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.

Results and recommendations

Abstract In the first phase of a collaborative study by the International Programme on Chemical Safety (PRCS), four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosurea (MNU), sodium azide (NaN 3 ) and maleic hydrazide (MH), and ethyl methanesulfonate (EMS) as a positive control were tested in four plant bioassays, namely the Arabidopsis embryo and chlorophyll mutation assay, the Tradescantia stamen hair assay (Trad-SH assay), the Tradescantia micronucleus assay (Trade-MCN), and the Vicia faba root tip assay. Seventeen laboratories from diverse regions of the world participated with four to six laboratories each using one plant assay. For the Arabidopsis assay, laboratories were in agreement with MNU and AG giving positive responses and NaN 3 giving a negative response. With the exception of one laboratory which reported MH as weakly mutagenic, no mutagenic response was reported for MH by the other laboratories. For the Vicia faba assay, all laboratories reported a positive response for MNU, AG, and MH, whereas two of the six laboratories reported a negative response for NaN 3 . For the Trad-SH assay, MH was reported as giving a positive response and a positive response was also observed for MNU with the exception of one laboratory. NaN 3 , which exhibited a relatively high degree of toxicity, elicited a positive response in three of the five laboratories. AG was found positive in only one of the two laboratories which tested this chemical. For the Trad-MCN assay, MNU and MH were reported as positive by all laboratories, while four out of five laboratories reported NaN 3 to be positive. Only one of three laboratories reported AG to be positive. The major sources of variability were identified and considered to be in the same range as found in similar studies on other test systems. Recommendations were made for minor changes in methodology and for initiating the second phase of this study.

Clastogenicity evaluation of seven chemicals commonly found at hazardous industrial waste sites

7 chemicals commonly found at the industrial waste sites were tested with the Tradescantia-Micronucleus (Trad-MCN) assay to evaluate their clastogenic potential. Chemicals selected from the US EPA Superfund Priority 1 list were: aldrin, arsenic trioxide, 1,2-benz[a, h]anthracene, dieldrin, heptachlor, lead tetraacetate and tetrachloroethylene. Results of repeated tests for clastogenicity yielded the minimum effective dose (MED) for clastogenicity of 0.44 ppm for lead tetraacetate, 1.88 ppm for heptaclor, 3.81 ppm for dieldrin and arsenic trioxide and 1,2-benz[a, h]anthracene yielded positive responses at the MED of 3.96 ppm and 12.50 ppm respectively. Aldrin and tetrachloroethylene were considered to be immiscible with water, and the tests yielded negative responses. Tetrachloroethylene in gaseous state was also used to treat the flower buds. Results of tetrachloroethylene vapor phase treatment yielded a positive response at the MED of 30 ppm/min after a 2-h exposure. 5 chemicals determined to be clastogens by this test were ranked according to their MED in the descending order of potency as follows: lead tetraacetate, heptachlor, dieldrin, arsenic trioxide and 1,2-benz[a, h]anthracene. Results of this study indicate that the Trad-MCN bioassay could be effectively utilized for assessing the potential clastogenicity of the chemicals commonly found at the industrial hazardous waste sites.

Arabidopsis assay for mutagenicity.

Four laboratories, two in the Czech Republic (Brno and Prague) and two in the CIS (Moscow and Duschanbe), participated in the International Programme on Chemical Safetys (IPCS) collaborative study to evaluate the utility of the most commonly used plant test systems, including the Arabidopsis thaliana assay, for assessing the mutagenic potential of environmental agents. Out of the five compounds evaluated in the Arabidopsis assay, three compounds, i.e., ethyl methanesulfonate, N-methyl-N-nitrosourea, and azidoglycerol, were reported to be mutagenic by all four participating laboratories. Sodium azide (NaN3) demonstrated a negative response in all four laboratories, whereas maleic hydrazide was reported to be weakly mutagenic by one laboratory and nonmutagenic by the other three laboratories.

Application of the tradescantia micronucleus assay for the genetic evaluation of chemical mixtures in soil and aqueous media

Genotoxic evaluations of arsenic trioxide, dieldrin, lead tetraacetate and their nine binary and one tertiary mixtures were performed using the Tradescantia micronucleus (Trad-MN) assay. The chemicals or their mixtures were either (1) mixed into soil, and chemical exposure to the target cells was through the roots of intact plants grown in the soil or (2) through plant cuttings in which the inflorescences received treatment by absorption through stem of an aqueous solution of the test chemicals. All three chemicals yielded clastogenic responses when tested in soil medium and only two of these i.e. arsenic trioxide and dieldrin were positive when plant cuttings were exposed to the test chemicals in the aqueous medium. The clastogenicity of the chemical mixtures was modified by the ratio of the individual chemical in a particular mixture and also by the medium in which these mixtures were tested.

Mutation Induction and Detection in Arabidopsis

Mutation is an infrequent event, and the stability of the various gene loci is quite different. Additional complexity appears when mutability is studied in different taxonomic groups displaying a variety of genome organization. Higher plants permit the study of mutation in three types of genomes: nuclear, plastidic and mitochondrial. In Arabidopsis the size of the nuclear genome is about 1. 3 × 1012 dalton whereas the chloroplast and the mitochondrial genomes are estimated to be 4 to 5 orders of magnitude smaller. The nuclear genome of Arabidopsis is much less redundant than that of other higher plants, thus mutation can be detected at an estimated 19,688 loci during embryonic development and the total number of loci with visible mutations appears to be 27,875.

Detection of chemically induced aneuploidy with plant test systems

Abstract The utility of plant test systems for detecting chemically induced aneuploidy was evaluated by using papers published in peer-reviewed journals. A total of 147 papers were provided to the group by the Environmental Mutagen Information Center. Based on the criteria established by the Gene-Tox Committee (Waters and Auletta, 1981), 22 papers were selected for in-depth review. Only those papers listing additional, missing, or lagging chromosomes in the meiotic or mitotic cells were included in this review. Although most plant test systems may be developed to utilize either mitotic or meiotic cells for cytogenetic analysis, only a few have been employed for this purpose. In this review, Allium cepa was found to be the most commonly used test system. Other species used less frequently were Vicia faba, Hordeum vulgare, Sorgham vulgare, and Pennisetum americanum. None of the plant test systems have been sufficiently utilized to warrant judgment for its sensitivity and specificity for detecting induced aneuploidy. A suggested protocol for detecting chromosomal malsegregation in meiotic or mitotic cells is presented. Further development and utilization of plant tissue culture techniques and morphological markers identifiable in the seedling stages is recommended for detecting chemically induced aneuploidy.

Evaluation of the Genotoxic Potential of Certain Pesticides Used in Pakistan

The mutagenicity of fifteen insecticides, five fungicides, four herbicides, and an acaricide commonly used in Pakistan was evaluated by employing thirteen short-term bioassays. The genetic endpoints used included point or gene mutation, primary DNA damage, and chromosomal effects. Initially, all pesticides were tested in a core battery of four in vitro bioassays. A carefully selected group among these chemicals was retested in higher level test systems to confirm the results obtained in the initial phase. Of the pesticides tested, only a small portion consistently displayed mutagenicity across test systems. The Saccharomyces cerevisiae bioassays detected mutagenicity for the largest number of pesticides. The Salmonellaces typhimurium strain, TA100, was able to detect genetic activity in all of the pesticides that produced positive results in this bioassay. The cytogenetic effects observed from the Vicia faba root assay were consistent with those obtained in mammalian cells in culture. All pesticides which displayed mutagenicity were not carcinogenic in animal bioassays but, in general, mutagenicity in a battery of short-term bioassays was a reliable indicator of the carcinogenic potential in animals. A simple test battery is proposed for evaluating the genetic potential of agricultural pesticides.