Biomedical subjects

M J Plewa

Publications and source records attributed to M J Plewa.

At least 19 recordsLinked to original sources

PubMed2001

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.

Animals↗

PubMed2000

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.

Alkylating Agents↗

PubMed2000

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.

Animals↗

PubMed1999

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.

Chromosome Aberrations↗

PubMed1999

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.

Alkylating Agents↗

PubMed1999

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.

Animals↗

PubMed1998

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.

Cell Division↗

PubMed1998

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.

Acetyltransferases↗

PubMed1998

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.

Animals↗

PubMed1998

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.

DNA Damage↗

PubMed1998

The International Comet Assay Workshop.

The Comet (single cell gel electrophoresis) assay primarily measures DNA strand breakage in single cells (Singh et al., 1988). Briefly, cells are suspended in low melting point agarose on a microscope slide. The slides are put in lysing buffer to allow the DNA to unwind and then in electrophoresis buffer. During electrophoresis the broken DNA moves towards the anode forming a Comet tail, with the greater the extent of damage, the greater the tail. Assays can be conducted under neutral or alkaline (> pH 13) conditions. Double-strand breaks are measured under neutral conditions and single-strand breaks under alkaline conditions, where abasic sites and other alkali-labile sites or intermediates in base or nucleotide excision repair can also be detected. There are several good reviews concerning the assay (McKelvey-Martin et al., 1993; Fairbairn et al., 1995; Tice, 1995).

Cell Extracts↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Acetoxyacetylaminofluorene↗

PubMed1997

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.

Biotransformation↗

PubMed1997

Involvement of nitroreductase and O-acetyltransferase on the mutagenicity of plant-activated benzidine and 4-aminobiphenyl.

Benzidine and 4-aminobiphenyl (4-ABP) are activated by intact plant cells and cell free TX1MX into mutagenic metabolites that induce frameshift and base pair substitution mutations in Salmonella typhimurium. The plant activation of these agents is plant peroxidase-mediated and bacterial O-acetyltransferase (OAT) dependent. TX1MX-activated benzidine and 4-ABP were analyzed with S. typhimurium frameshift tester strains, YG1021, YG1024, TA98, TA98NR, TA98/1,8-DNP6, MP219, and base pair substitution tester strains, YG1026, YG1029, TA100, TA100NR, TA100TN:OAT, and MP208. Concentration ranges for benzidine and 4-ABP were 1-50 microM and 0.1-1 mM, respectively. This study was conducted to determine if the plant-activation of benzidine and 4-ABP follows the prostaglandin H synthase-mediated activation pathway in mammals [Smith et al. (1992): Chem Res Toxicol 5;431-439]. In this model, benzidine is N-acetylated by S. typhimurium OAT. This acetylated product is a substrate for PHS and is converted into a 4-nitro product which is catalyzed by nitroreductase into a N-hydroxy intermediate. The pathway assigns a specific role for nitroreductase in the activation of benzidine. By employing S. typhimurium strains that express different levels of OAT and/or nitroreductase, we determined that the plant-activation of benzidine and 4-ABP has an absolute requirement of bacterial OAT activity for the induction of frameshift mutations at hisD3052 and is required for the optimal mutagenic response at hisG46. Nitroreductase also plays a role in the plant activation of these agents. The data suggest that the plant-activation of benzidine and 4-ABP generates at least two classes of proximal mutagenic intermediates. One class requires S. typhimurium OAT alone to be transformed into the ultimate mutagen and a second class requires both OAT and nitroreductase.

Acetyltransferases↗

PubMed1997

Plant-activation of the bicyclic aromatic amines benzidine and 4-aminobiphenyl.

Benzidine and 4-aminobiphenyl (4-ABP) are promutagenic bicyclic aromatic amines that are activated into frameshift and base pair substitution mutagens by plant systems. Using the plant cell/microbe coincubation assay, plant-activated benzidine from 0 to 50 microM induced a concentration-response in Salmonella typhimurium. At concentrations above 5 microM, plant-activated benzidine induced frameshift and base pair substitution mutations in the N- or O-acetyltransferase over-expressing strains, DJ460, YG1024, and YG1029. With plant-activated 4-ABP, concentrations above 250 microM induced a significant mutagenic response in strains YG1024 and YG1029. A tobacco cell-free mixture, TX1MX, activated benzidine and 4-ABP into mutagenic metabolites in S. typhimurium strains YG1024, YG1029, and DJ460. The mutagenic sensitivities of plant-activated benzidine and 4-ABP were the same with two different types of plant activation systems, TX1 suspension cells and TX1MX cell-free medium. The plant activation of these aromatic amines is mediated by tobacco cell peroxidase. Plant-activated benzidine and 4-ABP are converted into intermediates that serve as substrates for bacterial or humanacetylCoA: N-hydroxyarylamine N-acetyltransferase to generate the ultimate mutagenic products.

Acetyltransferases↗

PubMed1997

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.

Acetoxyacetylaminofluorene↗

PubMed1996

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.

Acetyltransferases↗