Te-Hsiu Ma

The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants.

The meristematic mitotic cells of plant roots are appropriate and efficient cytogenetic materials for the detection of clastogenicity of environmental pollutants, especially for in situ monitoring of water contaminants. Among several cytological endpoints in these fast dividing cells, such as chromosome/chromatid aberrations, sister-chromatid exchanges and micronuclei, the most effective and simplest indicator of cytological damage is micronucleus formation. Although the Allium cepa and Vicia faba root meristem micronucleus assays (Allium/Vicia root MCN) have been used in clastogenicity studies about 12 times by various authors in the last 25 years, there is no report on the comparison of the efficiency of these two plant systems and in different cell populations (meristem and F1) of the root tip as well as under adequate recovery duration. In order to maximize the efficiency of these bioassays, the current study was designed to compare the Allium and the Vicia root MCN assays on the basis of chromosome length, peak sensitivity of the mitotic cells, and the regions of the root tip where the MCN are formed. The total length of the 2n complement of Allium chromosomes is 14.4 microns and the total length of the 2n complement of Vicia is 9.32 microns. The peak sensitivity determined by serial fixation at 12-h intervals after 100 R of X-irradiation is 44 h. The slope of the X-ray dose-response curve of Allium roots derived from the meristematic regions was lower than that derived from cells in the F1 region. Higher efficiency was also demonstrated when the MCN frequencies were scored from the F1 cells in both Allium and Vicia treated with formaldehyde (FA), mitomycin C (MMC), and maleic hydrazide (MH). The results indicated that scoring of MCN frequencies from the F1 cell region of the root tip was more efficient than scoring from the meristematic region. The X-ray linear regression dose-response curves were established in both Allium and Vicia cell systems and the coefficients of correlations, slope values were used to verify the reliability and efficiency of these two plant cell systems. Based on the dose-response slope value of 0.894 for Allium and 0.643 for Vicia, the Allium root MCN was a more efficient test system. The greater sensitivity of the Allium roots is probably due to the greater total length of the diploid complement and the higher number of metacentric chromosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Genotoxic effects of heavy metals: Comparative investigation with plant bioassays

The potential use of micronucleus assays in plants for the detection of genotoxic effects of heavy‐metal ions was investigated. Three different plant systems were comparatively investigated in micronucleus with Tradescantia pollen mother cells (Trad MCN) and micronucleus tests with meristematic root tip cells of Allium cepa and Vicia faba (Allium/MCN).

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.

The international program on plant bioassays and the report of the follow-up study after the hands-on workshop in China.

Among the seven plant bioassays reviewed by the US Environmental Protection Agency (EPA) Gene-Tox program in 1980, the Allium/Vicia root tip chromosome aberration assay and the Tradescantia stamen hair mutation and micronucleus assays were adopted by the International Program on Plant Bioassays (IPPB) for monitoring or testing environmental pollutants. These assays are highly sensitive and capable of detecting mutagens, clastogens and carcinogens from the environment. They are effective in situ monitors. These bioassays were validated and their protocols were standardized through a program under the International Program on Chemical Safety (IPPB), the precursor of the IPPB program which currently is in operation under the auspices of the United Nations Environment Program (UNEP). Six different categories of environmental studies, ranging from wastewater, surface or ground water, soil samples, ambient air, pesticides, and radiation, were carried out and are reported in this special issue. The mission of the IPPB/UNEP is to use these bioassays to monitor or test environmental mutagens and clastogens in the air, water, and soil to safeguard the quality of these essential elements in life and to use these simple and clear indicators of pollution damage as the tool for environmental education for the general public. The published reports of the monitoring and testing results will establish the database for environmental conditions in a number of locations around the world. An international monitoring network for the detection of genotoxicity of environmental pollutants is being established under the auspices of UNEP to protect the global environment.

Tradescantia stamen hair mutation bioassay

The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100 micrograms/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45 micrograms/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80 micrograms/ml. NaN3, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN3 ranged between 3 and 80 micrograms/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well.

Micronuclei induced by X-rays and chemical mutagens in meiotic pollen mother cells of tradescantia: A promising mutagen test system

Abstract In Tradescantia, pollen mother cells of each of the buds of an inflorescence are synchronized at different meiotic stages, thus facilitating the treatment of the appropriate buds containing only, the mutagen-sensitive, prophase stage. Damage to chromosomes at early prophase can be determined by scoring the frequency of micronuclei (MCN) in tetrads 24–30 after treatment. This study demonstrated the high efficiency and versatility of this “MCN-in-Tetrad” test system for radiation and chemical mutagens. When inflorescences of the plant cuttings were exposed to 20 and 40 R of X-rays, an average of 22.8 and 66.6 MCN/100 tetrads were observed respectively. Liquid ethyl methanesulfonate (EMS), at 50 and 100 mM, absorbed through the stem, induced 13.2 and 15.2 MCN/100 tetrads respectively, while gaseous EMS (1000 ppm) induced 17.4 MCN/100 tetrads. Liquid sodium azide (NaN3), at 0.2 mM induced 10.1 MCN/100 tetrads, while 136 ppm of gaseous hydrazoic acid (HN3), which is the fume released from NaN3 reacted with acid, induced 21.2 MCN/100 tetrads. The control of each of the experimental groups yielded around 5 MCN/100 tetrads.

Genotoxicity of contaminated soil and shallow well water detected by plant bioassays.

Three major plant bioassays, i.e., the Allium root anaphase aberration (Allium-AA), the Tradescantia-micronucleus (Trad-MCN) and the Tradescantia stamen hair mutation (Trad-SHM) tests, were utilized in soil solutions or shallow well water samples to determine the degree of their genotoxicity. Shallow well water samples were collected from five different farms, and soil solutions were extracted with distilled water or dimethyl sulfoxide (DMSO) from pesticide-contaminated (metolachlor, atrazine, extrazine, and 2, 4-D) and pesticide-free soil samples. Genotoxicity was expressed in terms of anaphase aberration (AA) frequencies in the Allium-AA test, in terms of micronuclei frequencies in the Trad-MCN test, and in terms of pink mutation events in the Trad-SHM test. On average, results of Allium-AA tests showed a 2.78-3.01 fold increase in anaphase aberration frequencies in contaminated soil solution samples and well water samples as compared with the negative control. Results of Trad-MCN tests showed a 1.66-4.75 fold increase of MCN frequencies in contaminated soil solution samples and shallow well water samples as compared with the frequencies of the controls. Results of Trad-SHM tests showed a 2.7-2.86 fold increase of pink mutation events in the contaminated soil solution samples over that of the controls. Control groups of the Allium-AA tests had an average of 0.75/1000 anaphase figures, and control groups of the Trad-MCN tests had an average of 3.2 MCN/100 tetrads, while control groups of the Trad-SHM tests had an average of 1.4 mutation events/1000 hairs. In general, soil solutions of DMSO extracts showed higher genotoxicity than that of distilled water extracts. Among these three plant bioassays, the Trad-MCN test has the highest efficiency. The highest toxicity, based upon the Trad-MCN test results, was found in the pesticide contaminated soil samples from Monroes farm. Water samples from the Fountain Green/Bushnell area ranked second in genotoxicity.

In situ monitoring with the Tradescantia bioassays on the genotoxicity of gaseous emissions from a closed landfill site and an incinerator

A dual monitoring system composed of the Tradescantia-Micronucleus (Trad-MCN) and Tradescantia-Stamen-Hair-Mutation (Trad-SHM) bioassays was utilized to monitor directly the genotoxicity of the gaseous emission at a closed landfill site and around an incinerator. Four of the commonly emitted gaseous agents from the landfill flare pipes, i.e. toluene, ethylbenzene, trichloroethylene and ethyltoluene were also evaluated for their genotoxicity in the laboratory. The in situ monitoring trips (360 km one way) were carried out by transporting the plant cuttings in a clean air box or in an air-tight plastic bag to the site and exposing these test cuttings for 5-7 h. The exposed plant samples were examined for micronuclei frequencies or the pink mutation rate after the appropriate recovery periods (24 h for MCN, 7-11 days for SHM). A total of 20 monitoring trips were made to the landfill, and 8 to the nearby surroundings (100-500 m from the chimney) of the incinerator site in a two year period. The major findings of the Trad-MCN test on the clastogenicity of the gaseous emission from the flare pipe of the landfill site showed positive responses or toxic effects in 6 out of 20 trips, and that from the incinerator showed positive responses in 5 out of the 8 trips. These positive responses were closely associated with the weather, i.e. low wind velocity, high temperature and relative humidity, and especially the distance from the chimney of the incinerator. The MCN frequencies and mutation rates of the Elementary School site (E. Sch) which is about 200 m from the fence of the landfill site were mostly negative, except the test results of three trips. Trad-SHM tests on the mutagenicity of gaseous emissions from the flare pipe of the landfill showed 12 positive responses out of 20 trials and 2 positives out of 4 trials from the incinerator gaseous emissions. The average mutation rate from 20 Trad-SHM monitoring trips is positive when the ANOVA and Dunnetts t-statistic were applied to the consolidated data. There is a significant (0.01) difference between the lab control and the gas exposed groups, and between the field control and gas exposed groups. Results of the Trad-SHM test at the E. Sch. site were mostly negative except for one trip. In general, micronuclei frequencies and mutation rates of the field control groups were relatively higher than those of the lab controls. The Trad-MCN test on pure gases showed positive responses in all 3 repeated tests on toluene (50-892 ppm). The test results of ethylbenzene yielded positive responses at 172 ppm/min and 1549 ppm/min dosages and exhibited toxicity at higher concentrations. Trad-MCN tests on trichloroethylene and ethyltoluene yielded positive responses at around 100-200 ppm/min level. Three repeated Trad-SHM tests on toluene yielded no positive response at low concentrations (4.3-12.9 ppm).

Tradescantia-micronucleus (Trad-MCN) bioassay on clastogenicity of wastewater and in situ monitoring

The Tradescantia-micronucleus (Trad-MCN) bioassay was used to determine the clastogenicity of wastewater samples collected from the Arena canal which contains effluent from the industrial district Benito Juarez of the city of Queretaro, Mexico. Fifteen wastewater samples which were collected, in most cases, at bi-weekly intervals beginning in September 1986 through February 1988, after a 3-fold dilution were used to treat Tradescantia plant cuttings. The clastogenicity expressed in terms of micronucleus frequencies of treated groups (30 h of treatment without recovery time) was significantly (0.01) higher than that of the tapwater control groups. The Trad-MCN bioassay was also used for in situ monitoring of air pollutants for the clastogenicity at 3 sites near the industrial and residential areas (Flores Magon, Conalep and Bellas Artes) of the city of Queretaro. Fourteen monitoring trips were made to each of the 3 sites at monthly intervals beginning in May 1988 through June 1990. Seasonal variation of micronucleus frequencies was exhibited with the peak clastogenicities shown in May and June 1988, June 1989 and April 1990 at the three sites. Micronucleus frequencies of all the exposed groups at the Conalep site, a predominantly industrial area, were markedly higher than that of the laboratory control groups throughout the 2-year period.

Low level of SO2 enhanced chromatid aberrations in Tradescantia pollen tubes and seasonal variation of the aberration rates

Abstract Pollen tube cultures of Tradescantia paludosa Anders. Sax clone-3 were grown on microslides coated with lactose agar medium (0.02% colchicine). They were incubated in an air-tight moist chamber for 18 h to obtain the metaphase chromosomes of the pollen tube generative nuclei. SO2 treatments were applied at the beginning of the germination stage, 7–10 min after sowing the pollen. A total of about 0.075 ppm of SO2 was used for the entire incubation period. SO2 gas was either generated by the reaction between K2S2O5 and HCl inside the chamber or obtained from diluting the gas from commercial SO2 gas (Matheson Gas Products) of known concentration through a gas dilution system. Full-grown cultures fixed in Gates fluid and stained in Feulgen were made into permanent slides for aberration-rate determination. Year-round study of SO2 effect on chromosomes was made possible by using a GRO-LUX lamp (Sylvania) illuminated incubator, in winter, to promote mitotic activity of pollen culture. In these studies, SO2 enhanced chromatid aberration rates (average 46 breaks/100 cells) in the cultures grown from field or hot-house plants during 3 consecutive summers (1970–1972). There was a gradual decrease in the fall and a gradual increase in spring in both background and enhanced aberration rates. When summer pollen cultures from GRO-LUX lamp pre-conditioned (7–20 days) plants were treated with SO2 in the same chamber with those grown from summer hot-house plants, high rate (42 breaks/100 cells) of aberration was observed only in cultures from summer hot-house plants.