Isolating antigenotoxic components and cancer cell growth suppressors from agricultural by-products.

Commercial processing wastes or by-products of crops were found to be sources of antimutagens and human tumor cell growth suppressors. We developed a microplate method to measure genomic DNA damage in Chinese hamster ovary cells with a modified single cell gel electrophoresis (SCGE) assay. This allowed us to measure the repression of 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage by very small amounts of complex mixtures, fractions or individual chemicals isolated from agricultural by-products. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, repressed induced genomic DNA damage in mammalian cells. PCC was separated into a series of chemically defined fractions and two fractions (PCC70 and PCC100) repressed mutagen-induced damage. Of the isoflavones isolated from soybean fraction PCC70, daidzein expressed antigenotoxic activity, however, genistin and genistein enhanced DNA damage. An antigenotoxic response also was observed with a fraction isolated from corn distillate solids (CDS40). We developed a microplate assay to measure the suppression of the growth rate of human cancer cells in which the cytostatic/cytotoxic status at each concentration of the test sample was quantitatively determined. Genistein, genistin, daidzein and daidzin isolated from soybean fraction PCC70 expressed a wide range of growth suppression of HT-29 human colon cancer cells. The biological assays were integrated with, and directed, the separation and analytical chemistry component of this project. Compounds were purified from biologically active fractions and the structure of individual chemicals was determined with analytical HPLC and LC-mass spectroscopy (LC-MS). This research may lead to the isolation of novel chemoprotectants from agronomic commercial processing products and by-products.

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.

Characterization and antimutagenic activity of soybean saponins.

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.

Studies on the genotoxic effects of aminophenazines using two cellular models and three different methods.

This paper presents studies on the genotoxicity of two aminophenazines: 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP). The genotoxic activities of these compounds were evaluated with human lymphocytes using the alkaline single cell gel electrophoresis (SCGE) assay and two cytogenetic assays (chromosome aberrations (CA) and sister chromatid exchange (SCE) analysis). Results show that these chemicals elicited an increase in DNA and chromosomal damage under the studied ranges of concentration. Concentration-response curves were similar and there was a positive correlation between the damage observed at the DNA and chromosomal levels. DAP was more genotoxic than AHP and this agreed with the genotoxic potencies reported in bacterial systems.

Antimutagenic activity of chemical fractions isolated from a commercial soybean processing by-product.

Commercial products of agronomic crop plants may become a reliable and inexpensive source of phytonutrients, such as antimutagenic food supplements. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, was able to repress induced genomic DNA damage, whole cell clastogenicity, and point mutation in mammalian cells. In this paper we separated PCC into a series of chemically defined fractions and determined their ability to repress induced mutagenic damage in Chinese hamster lung cells, Chinese hamster ovary cells and human lymphocytes. Fraction PCC70 (PCC 70% methanol eluate) contained the flavonoids from PCC and daidzin and genistin repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage measured with single cell gel electrophoresis. Genistein, however, enhanced the genotoxic impact of 2AAAF. Fraction PCC100 (PCC 100% methanol eluate) had the greatest level of antigenotoxic activity against 2AAAF in CHO cells and repressed the genotoxic capacity of the dietary carcinogen 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) in human lymphocytes. These data indicate that commercial soybean products and by-products may be a source of chemoprotective food additives.

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.

The use of single cell gel electrophoresis and flow cytometry to identify antimutagens from commercial soybean by-products.

Single cell gel electrophoresis (alkaline Comet assay) and flow cytometric methods were combined into an assay that enables the analysis of direct DNA damage and longer-term whole cell clastogenicity in mammalian cells. We employed these techniques to analyze the antimutagenic activity of by-products of commercial soybean processing. At a concentration of 1 mg/ml, the soybean molasses by-product was found to repress 66% of the mutagenic capacity of the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) in Chinese hamster lung (CHL) cells. At a concentration of 50 microg/ml, fraction PCC (an ethanol extract of soybean molasses) repressed 70% of the genotoxic potency of 500 nM 2AAAF as measured by the Comet assay. Fraction PCC was also effective in protecting CHL cells from 2AAAF-induced clastogenic damage. Using a forward mutation assay in Chinese hamster ovary cells (line AS52), PCC protected the cells against 2AAAF-induced cytotoxicity and point mutation at a specific gene target. These data indicate that agronomic crops such as soybean may yield a wealth of commercially available antimutagenic agents that may be suitable as chemoprotective food supplements.

Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells.

The induction kinetics of genetic damage were measured in one clone of a mammalian cell line (CHO AS52) with three genotoxicity assays, the single cell gel electrophoresis (Comet) assay, laser beam flow cytometry and forward mutation. The first two assays allow for the rapid analysis of genotoxic damage in individual nuclei. The alkaline Comet assay detects DNA strand breaks, alkali-labile sites and incomplete excision repair sites. Flow cytometry measures chromosome damage that results in an unequal distribution of nuclear DNA in daughter cells. We calibrated these assays to compare acute DNA damage and longer term clastogenicity with forward mutation at the gpt locus using ethyl methanesulfonate (EMS). The EMS treatments were conducted in F12 medium for 2 h. AS52 cells carry a single functional gpt gene which provides for quantitation of gpt mutants by selecting for 6-thioguanine resistance. EMS induced a concentration-dependent response with median Comet tail moment values of 1.06 microns for the negative control and 64.6 microns with 20 mM. The coefficient of variation (CV) of the negative-control with flow cytometry was 2.33; the CV value increased to 4.87 in cells treated with 20 mM EMS, EMS (8 mM) induced a mutant frequency of 779.8 x 10(-6) at a relative survival of 64.4%. Genetic response factors were calculated and the data demonstrate that the induction kinetics of genetic damage as measured by the Comet assay (15.6) and flow cytometry (14.2) were more closely related than that determined for mutation induction (7.9). These three assays measure a wide spectrum of genetic events at the level of DNA, the gene and the chromosome and demonstrate the usefulness of the Comet assay and flow cytometry as two relatively rapid procedures to detect genotoxic damage in mammalian cells.

Analysis of mutagens with single cell gel electrophoresis, flow cytometry, and forward mutation assays in an isolated clone of Chinese hamster ovary cells.

We investigated the induction of DNA strand breaks in the single cell gel electrophoresis (SCGE or comet) assay with whole cell clastogenicity measured with flow cytometric analysis in cells from an isolated clone of the Chinese hamster ovary (CHO) AS52 cell line. Under identical treatment conditions the responses were compared with forward mutation at gpt using 2-acetoxyacetylaminofluorene (2AAAF), ultraviolet radiation (UV) and ethyl methanesulfonate (EMS). Cytotoxicity for each agent was evaluated in the SCGE and forward mutation assays. Forward mutation was 4-10-fold more sensitive than DNA strand breaks detected in the SCGE assay. For 2AAAF and EMS, the kinetics of the induction of genetic damage were similar for the three assays, although there were differences in sensitivity. With UV, the induction kinetics of gpt mutation differed from that expressed by SCGE and flow cytometric analysis. With the chemical mutagens 2AAAF and EMS, there was a high correlation between the SCGE assay and forward mutation and also between the SCGE assay and flow cytometry. There was no significant correlation between flow cytometry and forward mutation. With UV, only the SCGE assay and flow cytometry were correlated. Agent-specific variations in the intragenomic distribution of DNA damage for each mutagen was measured in the SCGE assay.

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.

Mutagenic analysis of 2,3-diaminophenazine and 2-amino-3-hydroxyphenazine in Salmonella strains expressing different levels of O-acetyltransferase with and without plant and mammalian activation.

2,3-Diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) are products generated from oxidative-type phenylenediamine hair dyes and are also present in pesticide formulations as contaminants. Earlier studies demonstrated that DAP and AHP were mutagenic in Salmonella typhimurium strains after mammalian microsomal activation. Plant systems can activate structurally similar arylamines. S. typhimurium strains have been developed that express elevated levels of acetyl-CoA: N-hydroxyarylamine O-acetyltransferase (OAT). O-acetyltransferase expression is necessary for the generation of the ultimate arylamine promutagen after plant activation. A number of arylamines including 2-aminofluorene, benzidine and 4-aminobiphenyl were activated by plant cells into mutagens in the OAT over-expressing S. typhimurium strain, YG1024. The objectives of this research were to examine the mutagenicity of DAP and AHP with mammalian or plant activation in Salmonella strains with different acetyltransferase activities. The hypothesis tested was whether and to what degree a metabolite of DAP or AHP could serve as a substrate for bacterial O-acetyltransferase and induce mutation in Salmonella. DAP and AHP without activation induced both frameshift and base pair substitution mutations in S. typhimurium strains that exhibited elevated levels of O-acetyltransferase activity. The mutagenicity of DAP and AHP were greatly enhanced with mammalian hepatic microsomal activation resulting in a preferential induction of frameshift mutations. With the hisD3052 allele as the gene target, S9-activated DAP induced frameshift mutations in YG1024 and TA98 as well as the OAT deficient strain TA98/1,8-DNP6. S9-activated AHP induced mutation only in the OAT over-expressing strain, YG1024. With the hisG46 allele, O-acetyltransferase activity was necessary for the metabolism of DAP and AHP to products that induce base pair substitution mutations. An intriguing finding of this work was the antimutagenic capacity of TX1MX, a plant cell-free activation mixture. TX1MX repressed the mutagenic activity of DAP and AHP at frameshift and base pair substitution mutation targets.

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.

Genotoxicity of m-phenylenediamine and 2-aminofluorene in Salmonella typhimurium and human lymphocytes with and without plant activation.

The promutagenic arylamines, m-phenylenediamine (mPDA) and 2-aminofluorene (2-AF), were evaluated for their genotoxicity in Salmonella typhimurium strain YG1024 and in human lymphocytes. These agents were assayed with and without TX1MX plant activation mix. Both arylamines without activation were refractory in S. typhimurium, demonstrating that plant activation was required for the generation of their ultimate mutagenic metabolites. However, using the alkaline single-cell gel/Comet assay, both mPDA and 2-AF directly induced DNA damage in human lymphocytes. This effect was reduced when the human cells were treated with the arylamine plus TX1MX. mPDA with or without plant activation was not toxic to the exposed cells. However, at concentrations over 80 microM, 2-AF was toxic to lymphocytes. This toxic response was eliminated by incubation with TX1MX. mPDA and 2-AF were plant-activated into mutagens for S. typhimurium. However, these plant-activated products had a reduced genotoxic potency in human lymphocytes.

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.

Comparative mutagenicity of plant-activated aromatic amines using Salmonella strains with different acetyltransferase activities.

Plant systems can activate aromatic amines into mutagens. In the plant cell/microbe coincubation assay, we earlier demonstrated that 2-aminofluorene and m-phenylenediamine were activated by plant cells into mutagens with reversion at the hisD3052 allele in Salmonella typhimurium strain TA98 as the genetic endpoint. New derivatives have been developed which possess elevated levels of acetyl-CoA:N-hydroxyarylamine O-acetyltransferase which are very sensitive to N-hydroxylated amines [Watanabe et al., 1990: Mutat Res 234:337-348]. The objectives of this research were to examine Salmonella strains with different acetyltransferase activities in the plant cell/microbe coincubation assay with a series of structurally related aromatic amines. The hypothesis tested was whether and to what degree a plant-activated metabolite of these aromatic amines could serve as a substrate for bacterial O-acetyltransferase and induce mutation in Salmonella. Every aromatic amine examined was activated by plant cells with YG1024 (the strain with elevated O-acetyltransferase activity) as the genetic indicator organism. The rank order of the mutagenic responses of YG1024 to the plant-activated aromatic amines was 2-aminofluorene > benzidine > m-phenylenediamine > 4-aminobiphenyl > 2,4-diaminotoluene > 2-naphthylamine. This rank order was expressed by TA98 but to a much lower degree. There was a very slight mutagenic response observed in TA98/1,8-DNP6 (a strain lacking O-acetyltransferase activity) with the plant-activated metabolites of benzidine and 4-aminobiphenyl. We conclude that the plant-activated aromatic amines are substrates for bacterial O-acetyltransferases.