o-Phenylenediamine-induced DNA damage and mutagenicity in tobacco seedlings is light-dependent.

Of the three isomers of the aromatic amine phenylenediamine (PDA), only o-PDA, but not m- and p-PDA, induced DNA damage (as measured by the Comet assay), and somatic mutations in the leaves of the chlorophyll-deficient tester strain Nicotiana tabacum var. xanthi. With increasing light intensity (0, 30, 80 or 140 micromol m(-2)s(-1) photosynthetic photon fluence rate) during a 72h mutagenic treatment of tobacco seedlings, o-PDA-induced DNA damage and the yield of somatic mutations were significantly increased. The peroxidase inhibitor diethyldithiocarbamate (DEDTC) repressed o-PDA-induced DNA damage. The effect of light is caused by the light-dependent increase of peroxidase activity and the accumulation of hydrogen peroxide, which participate in the metabolic activation of the promutagen o-PDA to mutagenic product(s). In contrast, DNA damage induced by the direct-acting alkylating mutagen ethyl methanesulphonate was the same whether treatment was in the light or in the dark, and was not repressed by the peroxidase inhibitor DEDTC.

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings.

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.

A comparison of DNA repair using the comet assay in tobacco seedlings after exposure to alkylating agents or ionizing radiation.

We employed single cell gel electrophoresis to analyze the kinetics of DNA repair in nuclei isolated from tobacco plants exposed to ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU) and gamma-radiation. DNA repair was measured as the reduction of the tail moment values as a function of time after the mutagen treatment ended. DNA damage in leaf nuclei of EMS-or ENU-treated tobacco plants persisted over a 72h recovery period. However, a reduction of the SCGE tail moment values in nuclei isolated from leaves was observed over a 4-week period of recovery. Newly emerged leaves expressed a lower level of DNA damage due to more efficient repair and/or dilution of initial DNA lesions during cell division. After 24h recovery, leaf nuclei from cells exposed to 20 or 40Gy of gamma-radiation expressed complete DNA repair. These data indicate that DNA lesions induced by alkylating agents are not readily repaired and persist beyond 4 weeks. Enzymes necessary to repair gamma-induced DNA lesions are fully functional in non-replicating leaf cells and single and double strand breaks are rapidly repaired.

Maleic hydrazide induces genotoxic effects but no DNA damage detectable by the comet assay in tobacco and field beans.

The plant growth regulator and herbicide maleic hydrazide (MH) induced a high frequency of somatic mutations in leaves of tobacco (Nicotiana tabacum var. xanthi) and a high yield of chromosome aberrations in roots of field beans (Vicia faba, karyotype ACB). In contrast, no significant increase in MH-induced DNA damage, as measured by the Comet assay, could be demonstrated in either plant species. The absence of DNA migration induced by MH was not effected in tobacco by either pH of the MH solution, the sampling time after MH treatment or continuous MH treatment for 14 days. To our knowledge, MH represents the first agent which has proved to be highly mutagenic and clastogenic but does not cause DNA damage as measured by the Comet assay in the same experimental system.

Comparison of DNA damage in plants as measured by single cell gel electrophoresis and somatic leaf mutations induced by monofunctional alkylating agents.

The use of single cell gel electrophoresis (SCGE) has recently been applied to plant systems. We optimized the experimental conditions for SCGE analysis using nuclei isolated from different tissues of intact plants. Concentration-response curves of genomic DNA migration were analyzed in intact plants treated with the monofunctional alkylating agents ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), and N-methyl-N-nitrosourea (MNU). These data were used to calibrate SCGE tail moment values to induced somatic mutation in plant leaves. We used a genotoxicity index to compare genomic DNA damage and the induction of somatic mutation in the leaf tissues. The rank order of the genotoxic potency of these alkylating agents assayed by SCGE was MNU >> MMS > ENU > EMS. The rank order for the mutagenic potency of these agents was MNU >> ENU congruent with MMS > EMS. The data demonstrate the utility of SCGE analysis in plant systems. The use of SCGE will permit a larger range of plants for use as in situ environmental monitors. Also, this approach may be used to search for crop plant germplasm accessions with enhanced genomic stability. We investigated whether the intragenomic distributions of DNA damage induced by these alkylating agents were uniform and random. When a plot of the ratio of the %tail DNA and tail length versus the concentration of the test mutagen was generated, the induced SCGE data deviated from a random distribution of genomic DNA damage.

Monitoring the genotoxicity of soil extracts from two heavily polluted sites in Prague using the Tradescantia stamen hair and micronucleus (MNC) assays.

We have taken soil samples from two sites in Prague, the capital of the Czech Republic, that are heavily polluted by motor vehicles. As a negative control, soil samples from a recreational site in Prague with no motor vehicle traffic were used. Soil samples from these sites were extracted with water or 5% dimethylsulphoxide (DMSO) for 24 h and cuttings of Tradescantia clone 4430 were immersed for 12 h at 25 degrees C in the extraction solutions. As a positive control Tradescantia plants have been treated with the promutagenic arylamine o-phenylenediamine at the same treatment conditions. None of the tested soil extractions significantly increased the frequency of somatic mutations in the stamen hair assay. By contrast, a 5% DMSO soil extract from one of the tested sites (entrance of the Letná tunnel) significantly increased the frequency of micronuclei (MNC) in the pollen mother tetrad cells. A repetition of the treatment 14 days later also resulted in an increase in the frequency of MNC, however the increase was not statistically significant. This study was conducted for the International Programme on Plant Bioassays.

Pentachlorophenol-mediated mutagenic synergy with aromatic amines in Salmonella typhimurium.

Pentachlorophenol (PCP), a widely used pesticide, enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) when assayed with specific Salmonella typhimurium strains. With 2AF the mutagenic synergy was observed in strains YG1024, TA1538, and MP153. With 2AAAF the PCP-mediated synergy was observed with these strains and with strain TA98/1,8-DNP6. The synergy was dependent upon the presence of an activated N-acetoxy functional group and was only expressed at the hisD3052 allele and not at the hisG46 allele. Spectrophotometric analysis demonstrated that the rate of degradation of 2AAAF was reduced in the presence of PCP in phosphate buffer or with S. typhimurium cytosol and thus PCP may be affecting the stability of the N-acetoxy group of activated aromatic amines.

Single cell gel electrophoresis analysis of genomic damage induced by ethyl methanesulfonate in cultured tobacco cells.

A procedure for employing cultured tobacco cells (line TX1) in the SCGE assay was developed. The effect on DNA migration was studied in control and EMS-treated cells at different stages in their growth curve. The experimental parameters of treatment time and the unwinding time were analyzed in TX1 cells. With EMS in a concentration range from 0 to 30 mM the average median (+/-S.E.) tail moments ranged from 2.71+/-0.24 microm for the negative controls and increased in a direct concentration dependent manner to 57.89+/-4.13 for cells treated with 30 mM EMS. Nuclei isolated from TX1 cells and treated with EMS had a similar sensitivity as TX1 cells after EMS treatment. The plant cells express similar concentration-response curves for EMS as reported with mammalian (CHO) cells. This plant cell SCGE assay may prove to be a useful tool for the study of agricultural chemicals in specific plant cell types, to compare the response of mutagens in plant and animal cells and for basic research in genetic toxicology and DNA repair in plants.

Induction of somatic DNA damage as measured by single cell gel electrophoresis and point mutation in leaves of tobacco plants.

The induction and measurement of DNA damage in nuclei of plant tissues is a new area of study with the alkaline single cell gel electrophoresis/comet assay. Methods to isolate plant cell nuclei cause high levels of DNA damage which are detected by the comet assay. We developed a method to isolate nuclei from leaf tissue of Nicotiana tabacum (a1+/a1; a2+/a2) in a modified Sörensen buffer that resulted in constant, low tail moment values for the negative controls. After treating intact tobacco plants with 1-8 mM ethyl methanesulfonate (EMS) we obtained a direct concentration-response with an average median tail moment of 65.9+/-4.4 micro(m) for plants exposed to the highest EMS concentration as compared to the median control tail moment value of 4.1+/-0.8. We found that the highest resolution was obtained with electrophoretic conditions of 0.74 V/cm at 300 mA for 20 min. Multiple leaves could be analyzed per plant within each treatment group and the tail moments were not significantly different. Tobacco seedlings were treated with EMS in the same manner as used for the comet assay and mutations were induced in the leaf primordia. The mean mutant frequency for the control was 1.46+/-0.20 mutant sectors/leaf. The mutant frequency increased in a concentration dependent manner; the mutant frequency induced by 8 mM EMS was 37.89+/-2.37 mutant sectors/leaf. The comet tail moment values and the leaf mutant frequency were highly correlated (r=0.98). The genetic response factor was calculated by the ratio of the difference in the response within the linear portion of each concentration-response curve divided by the slope of the curve. The genetic response factor for the tail moment was 7.82 while the value for mutation induction was 7.76. In this paper we describe a sensitive method with high resolution to apply the alkaline comet assay to plant leaves. The comet assay response was compared to that of induced point mutation. With this sensitive method for nuclei isolation from plant leaves, the alkaline SCGE assay could be incorporated into in situ plant environmental monitoring.

Characterization of a macromolecular matrix isolated from tobacco suspension cell cultures and its role in the activation of promutagenic m-phenylenediamine.

The medium recovered from the tobacco cell suspension cultures (TX1MX) activated the promutagenic aromatic amine m-phenylenediamine (mPDA) and a macromolecular complex (gel) responsible for the arylamine activation was isolated from the medium. The gel formation and the role of the gel components in the plant activation of mPDA to products mutagenic in S. typhimurium YG1024 were studied. The activation of mPDA was caused by the peroxidases present in TX1MX. We demonstrated an association of the peroxidase activity and gel pectins. Formation of a stable mutagenic association of mPDA with the macromolecular material was observed. The data indicate that the gel isolated from TX1MX is the macromolecular component of the arylamine conjugate proposed in earlier work.

Mutagenic synergy between paraoxon and mammalian or plant-activated aromatic amines.

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester (OP) insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy with paraoxon and plant-activated m-phenylenediamine or with direct-acting 2-acetoxyacetylaminofluorene in Salmonella typhimurium cells [Gichner T et al. (1996): Environ Mol Mutagen 27; 59-66]. In the present study, mammalian-activated m-phenylenediamine, o-phenylenediamine, p-phenylenediamine, benzidine, 2,3-diaminophenazine or 2-aminofluorene, as well as plant-activated benzidine or 2-aminofluorene expressed an elevated mutagenic potency when assayed with S. typhimurium strain YG1024 in the presence of paraoxon. Under non-toxic conditions, paraoxon amplified the S. typhimurium mutant yield induced by these aromatic amines between 1.9-fold and 8.4-fold. Spectrophotometric analysis demonstrated that the rate of degradation of 2-acetoxyacetylaminofluorene was not significantly different in phosphate buffer with or without paraoxon or with S. typhimurium cytosol with or without paraoxon. Also paraoxon-mediated mutagenic synergy does not appear to be due to a direct reaction with aromatic amines. Mutagenic synergy between aromatic amines and OP oxon products may be a cause of concern because people are chronically exposed to environmental and dietary aromatic amines, and a significant segment of the U.S. population tested positive for OP insecticide residues.

Plant activation and its role in environmental mutagenesis and antimutagenesis.

This paper reviews the use of in vitro and in vivo antimutagenicity studies that determined the role of plant peroxidases in the activation of arylamine promutagens. New information presented here suggests a model in which tobacco cell peroxidases exuded into the culture medium undergo a maturation process affecting their capacity to activate arylamine promutagens. Tobacco cell peroxidases are present in medium recovered from stationary phase cells and are associated with a fraction that sediments at 12000 x g. These peroxidases have a greater capacity to activate arylamines than do peroxidases present in the supernatant fluid. These data suggest that the plant activation of arylamines into products that are mutagenic in Salmonella typhimurium may be intimately involved in the process of lignification.

Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2-acetoxyacetylaminofluorene.

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy when paraoxon was incubated with plant-activated m-phenylenediamine (mPDA) or with direct-acting 2-acetoxyacetylaminofluorene (2AAAF) and S. typhimurium tester strains. Using non-toxic concentrations of plant-activated mPDA and paraoxon a 10-fold increase in the mutant yield of S. typhimurium was observed. The mutagenicity of the plant-activated mPDA product required that O-acetyltransferase (OAT) be expressed by the S. typhimurium tester strain. However, the paraoxon-dependent mutagenic synergy was observed using the direct-acting arylamine metabolite, 2AAAF, with strains YG1024, TA98 and TA98/1,8-DNP6 regardless of their OAT activity. This mutagenic synergy is dependent upon the presence of an activated acetylated form of the arylamine. The data presented here demonstrate that this mutagenic synergy is limited to paraoxon and not to the parent compound (parathion) or to a major metabolite of parathion (p-nitrophenol).

Metabolic activation of m-phenylenediamine to products mutagenic in Salmonella typhimurium by medium isolated from tobacco suspension cell cultures.

Both tobacco cells in suspension and the medium recovered from the suspension cultures (TX1MX) activated the aromatic amine m-phenylenediamine (m-PDA) into a product that was mutagenic in Salmonella typhimurium TA98 and YG1024. Medium recovered from stationary-phase tobacco cell cultures exhibited the highest level of m-PDA activation. No cytochrome P-450 was detected in the activating medium. A high molecular weight matrix having the highest m-PDA activating capacity and associated with a substantial fraction of the total peroxidase activity was isolated by Centricon-100 ultrafiltration of TX1MX. The data suggest that the peroxidases present in the recovered cell culture medium or in the high molecular weight matrix are responsible for the plant activation of m-PDA.

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 Safety's (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.

Antimutagenicity of three isomers of aminobenzoic acid in Salmonella typhimurium.

The m-, o- and p-isomers of aminobenzoic acid (ABA) repressed the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium TA100. Their antimutagenic potency was in the order of o-ABA > m-ABA > p-ABA. The mechanism of this antimutagenicity is ascribed mainly to the decomposition of MNNG induced by the aminobenzoic acid isomers outside or within the bacterial cells. The inhibition of plant cell peroxidases and bacterial acetyltransferases that are required for the plant activation of 2-aminofluorene (2-AF) to mutagenic product(s) may participate in the repression of 2-AF mutagenesis by the aminobenzoic acids in S. typhimurium strain YG1024. The aminobenzoic acid isomers exhibited no inhibitory effects towards the direct-acting agent 2-acetoxy-2-acetylaminofluorene, the stable diacetylated metabolic product of 2-AF.

An E. coli ada transgenic clone of Nicotiana tabacum var. Xanthi has increased sensitivity to the mutagenic action of alkylating agents, maleic hydrazide and gamma-rays.

Two transgenic clones X3 and X15 of Nicotiana tabacum var. Xanthi, heterozygous in two genes (a1 and a2) for chloroplast differentiation and transformed with the E. coli DNA repair gene ada cloned downstream from the 1' direction of the dual mas promoter, differed in the expression of the ada gene, in the number of copies of integrated T-DNA and in the response to the mutagenic action of alkylating and non-alkylating agents. The X3 genome contained four copies and the X15 genome one copy of T-DNA, nevertheless the expression of the ada gene, measured by the activity of O6-alkylguanine DNA alkyltransferase (ATase), was about six times higher in X15 than in X3. ATase activity in both clones was highest in extracts from callus whereas very low (X15) or no (X3) activity was detected in leaf extracts. This may explain the lack of difference between X15 and non-transformed tobacco (NTX) in the frequency of N-methyl-N-nitrosourea (MNU)-induced somatic mutations in leaves. In contrast, the frequency of somatic mutations in X3 was about 2-5 times higher than in NTX and X15 after the same doses of MNU, methyl methanesulfonate, maleic hydrazide and gamma-rays. Alteration of plant gene(s) essential in mutation pathway(s) by insertion of T-DNA or by somaclonal variation may explain the higher sensitivity of the X3 clone.

Induction of somatic mutations in Tradescantia clone 4430 by three phenylenediamine isomers and the antimutagenic mechanisms of diethyldithiocarbamate and ammonium meta-vanadate.

Three isomers of the promutagen phenylenediamine at mM concentrations were plant-activated and induced mutation in stamen hairs of Tradescantia clone 4430. The rank order of the mutagenicity of the isomers was: o-phenylenediamine > m-phenylenediamine > p-phenylenediamine with corresponding mutagenic potencies of 5.60, 1.43, and 0.46 mutant stamen hair cells/mumole, respectively. Diethyldithiocarbamate (DEDTC) and ammonium meta-vanadate (vanadate) repressed the mutagenic activity of o-phenylenediamine (o-PDA) in intact plants. Based on inhibition kinetics and reaction rates, the mechanism of DEDTC antimutagenicity was attributed to the inhibition of peroxidases that are required in the plant activation of o-PDA to mutagenic product(s). Spectrophotometric measurements of equimolar concentrations of o-PDA and vanadate demonstrated that the antimutagenic property of vanadate was mainly due to its reactivity with o-PDA.

Inhibitory effects of acetaminophen, 7,8-benzoflavone and methimazole towards N-nitrosodimethylamine mutagenesis in Arabidopsis thaliana.

The metabolic inhibitors acetaminophen, 7,8-benzoflavone, and methimazole significantly reduced the mutagenicity of the promutagen N-nitrosodimethylamine in the higher plant Arabidopsis thaliana. In contrast, these metabolic inhibitors had no effect on the mutagenicity of the direct-acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine.

Ozone is not mutagenic in the Tradescantia and tobacco mutagenicity assays.

Ozone fumigation of a double heterozygous chlorophyll mutant Nicotiana tabacum var. xanthi n.c. with concentrations up to 300 nl/l and of a heterozygous Tradescantia clone 4430 with concentrations up to 800 nl/l did not increase the frequency of somatic mutations above the spontaneous levels. However, ozone fumigation at these concentrations led to distinct physiological damage to plant tissues.