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.     

The use of yeast cultures for the detection of environmental mutagens using a fluctuation test.

A microbial fluctuation test, modified for the detection of environmental mutagens has been evaluated using a number of strains of the yeast Saccharomyces cerevisiae. Auxotrophic diploid cultures of yeast which produce prototrophic colonies by both mitotic gene conversion and mutation have been extensively utilized for the detection and evaluation of chemicals showing genetic activity. A number of the yeast strains utilized were shown to be suitable for use in the fluctuation test although the time scales of the experiments were considerably extended (up to 16 days) compared to those involving bacteria. The yeast strains respond to doses of mutagens at least a 100-fold lower than that required in a conventional short exposure treat and plate experiment. In experiments involving the induction of mitotic gene conversion at the tryptophan-5 and histidine-4 loci in the fluctuation test significant increases in prototrophic cells were produced in the presence of the insecticide Lindex (0.05 microng/ml), the preservative Thiomersal (0.0001 microng/ml), a mahogany hair dye (0.01 microng/ml), the herbicide Paraquat (0.02 microng/ml) and the alkylating agent ethyl methane sulphonate (0.1 microng/ml). The results demonstrate that the fluctuation test provides an extremely sensitive assay for the detection of chemicals which show genetic activity in yeast at non-toxic concentrations.     

Use of the spot,plate and suspension test systems for the detection of the mutagenicity of environmental agents and chemical carcinogens in Neurospora crassa

N23 and N24, selected from hundreds of ad-3 mutants, have been used as testers for the spot, plate and suspension tests in Neurospora crassa. These two testers are highly sensitive to mutagens and are revertible by a specific group of chemicals. N23 can be reverted from an adenine-dependence to adenine-independence by agents which cause base-pair substitutions whereas N24 can be reverted by frameshift mutagens. Studies described here show that spot, plate and suspension tests using testers N23 and N24 are satisfactory substitutes for the ad-3 forward-mutation system for screening the mutagenic activity of environmental agents and chemical carcinogens in N. crassa.     

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.     

Use of the spot, plate and suspension test systems for the detection of the mutagenicity of environmental agents and chemical carcinogens in Neurospora crassa.

N23 and N24, selected from hundreds of ad-3 mutants, have been used as testers for the spot, plate and suspension tests in Neurospora crassa. These two testers are highly sensitive to mutagens and are revertible by a specific group of chemicals. N23 can be reverted from an adenine-dependence to adenine-independence by agents which cause base-pair substitutions whereas N24 can be reverted by frameshift mutagens. Studies described here show that spot, plate and suspension tests using testers N23 and N24 are satisfactory substitutes for the ad-3 forward-mutation system for screening the mutagenic activity of environmental agents and chemical carcinogens in N. crassa.     

Development of an in situ microbial mutagenicity test system for airborne workplace mutagens: laboratory evaluation

A simple on-site Salmonella mutagenicity test system for the detection of airborne mutagens in the workplace is being developed. The system permits entrapment of mutagenic airborne particles and vapors by impinging unfiltered ambient air into trapping medium containing bacterial tester cells. The trapping device consists mainly of a pump, an impinger and a cyclone. The impinging air flow generated by the pump is approximately 3 1/min. New Salmonella typhimurium testers which are resistant to streptomycin (Str) and 8-azaguanine (AG) were derived from the Ames testers TA98 and TA100 and the arabinose-resistant tester SV50, and were used as mutation indicators. Microbial contamination was sufficiently controlled by addition of ampicillin, Str, AG, and cycloheximide to the trapping and plating media. New tester strains retained a high mutagenic sensitivity from their parent strains. Laboratory studies with volatile mutagens (methyl methanesulfonate, ethyl methanesulfonate, and dimethylnitrosamine) showed that the vapor trapping of this system is promising. The study with suspended silica particles coated with a known mutagen (2,4,7-trinitro-9-fluorenone) indicated that the particle trapping of the system is satisfactory. Incorporation of metabolic activation into the trapping medium by confining S9 mix and tester cells in dialysis tubing enabled this system to detect promutagens. This in situ system may be useful for mutagenic monitoring in the workplace.     

Development of immunochromatographic test systems for express detection of plant viruses

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rodshaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 μg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.     

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.     

The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: the National Toxicology Program Data Base

A portion of the U.S. National Toxicology Program (NTP) Salmonella typhimurium mutagenicity data base was analyzed by CASE, an artificial intelligence SAR system. CASE identified 13 structural determinants which, with a high probability (p less than or equal to 0.05) predicted the likelihood of mutagenicity of the 243 chemicals in the data base (sensitivity = 0.989; specificity = 0.950) as well as of chemicals not included in the data base. CASE also identified an additional set of structures which were highly predictive of mutagenic potency (sensitivity = 0.949; specificity = 1.00). Even though there is little overlap among the chemicals included in the NTP and Gene-Tox Salmonella data bases, CASE found significant similarities between the structural determinants of the mutagenicity in the two data bases, thereby validating the analyses and indicating a commonality in the structural basis of mutagenicity.     

A critical assessment of the OECD collaborative study to validate the uterotrophic assay for the detection of oestrogenic and anti-oestrogenic chemicals

The design and execution of a recently completed validation study on the rat uterotrophic assay for detecting oestrogens and anti-oestrogens, managed by the OECD, are critically assessed with respect to internationally agreed criteria for the validation of new in vitro and in vivo toxicity test methods. It is concluded that, while the design of the study did not take account of several important criteria for validation, the uterotrophic assay appears to reliably detect the strong and weak oestrogenic substances used in the study, which act via binding to the oestrogen receptor in vivo. However, the reliability of the assay has not been substantiated for detecting anti-oestrogens that act as antagonists, due to the involvement of an insufficient number of experiments and test chemicals. Moreover, the data do not permit an assessment of the accuracy of the prediction of oestrogenicity, and the protocols have not been sufficiently optimised with regard to controlling variables. This problem has been exacerbated by a wish to introduce as much flexibility as possible into the protocols during the formal validation phase of the study, rather than during a separate prevalidation stage. In addition, the choice between surgically treated and/or immature animals, and details of housing and husbandry conditions that are necessary for increasing the sensitivity and efficiency of the assay, need to be clarified. The assay also lacks a well-defined prediction model by which the overall relevance of the data to toxicity, and especially to human hazard, can be assessed, and no performance criteria have been established. The results of this analysis of the study indicate that it would be premature to produce an OECD test guideline for the uterotrophic assay at this time, before some of the above issues have been satisfactorily resolved.