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Biomedical subjects

M J Plewa

Publications and source records attributed to M J Plewa.

At least 19 recordsLinked to original sources
PubMed1995

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.

Adult↗
PubMed1995

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.

Biotransformation↗
PubMed1995

Analysis of spontaneous frameshift mutations in REV1 and rev1-1 strains of Saccharomyces cerevisiae.

Frameshift mutations occur by a number of mechanisms. To better understand the nature of these mechanisms, we determined the DNA sequence changes of 232 independent, spontaneous frameshift mutations in the HIS4 gene of REV1 and rev1-1 strains of Saccharomyces cerevisiae. All frameshift mutants were selected based on their ability to revert the +1 frameshift mutation his4-38. DNA sequence information was recovered using two approaches-the double-strand gap repair of plasmid pMP4, and the polymerase chain reaction (PCR). Using these techniques, saturated mutation spectra for the spontaneous reversion of his4-38 were generated. The most frequently occurring mutational events in both strains were -1 frameshifts, but +2 frameshifts, larger deletions, larger insertions and more complex mutations were also observed. Between the REV1 and rev1-1 strains, we noticed a significant difference in the distribution of -1 frameshift mutations. In addition, while for -1 frameshift events there was no significant difference between the reversion spectra determined by double-strand gap repair or PCR, there was a surprisingly significant difference between the types of frameshift mutations recovered by double-strand gap repair (only -1 frameshifts and one +2 frameshift), and those recovered using PCR (-1 frameshifts, +2 frameshifts, larger deletions and insertions, and more complex mutations). This difference may reflect a selectional mechanism inherent in double-strand break repair that avoids chromosomal sequences which include complex alterations.

Base Sequence↗
PubMed1994

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.

Acetoxyacetylaminofluorene↗
PubMed1994

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.

Ditiocarb↗
PubMed1993

Characterization of stable high molecular weight mutagenic product(s) of plant-activated m-phenylenediamine.

Monocyclic aromatic amines are environmental contaminants and many are promutagens and procarcinogens. Cultured tobacco cells, strain TX1, activated m-phenylenediamine into a frameshift mutagen that reverted the hisD3052 allele in Salmonella typhimurium strains TA98 and YG1024. However, the plant-activated products were refractory in strain TA98/1,8-DNP6. This indicated that these plant-activated products were substrates for bacterial acetyl-CoA: N-hydroxyarylamine O-acetyltransferase. A stable, high molecular weight (> 300 kDa) proximal mutagen was isolated by molecular ultrafiltration membranes. No parent compound was associated with the isolated mutagenic fraction. The high molecular weight fraction induced mutation in S. typhimurium strains TA98, YG1021 and YG1024. From these data we propose a model for the plant-activation of aromatic amine promutagens.

Acetyltransferases↗
PubMed1992

Analysis of the genotoxicity of municipal solid waste incinerator ash.

Combined bottom and fly ash obtained from a Chicago, IL, municipal solid waste incinerator (MSWI) was extracted with organic solvents, water or acidified water. The mean amounts of organic material isolated from each extraction procedure were 688.2, 91.8 and 167.7 micrograms/g MSWI ash. These extracts were evaluated for toxicity and mutagenicity in Salmonella typhimurium strains TA98 and TA100. We developed and calibrated a micropreincubation assay to evaluate small concentrations of the organic extracts. No direct-acting mutagens were found, however the acid-treated aqueous extracts were toxic. Materials isolated with methylene chloride methanol were mutagenic after hepatic microsomal activation (S9). The mutagenic potencies of the organic extract normalized to a per gram ash basis was the induction of 103.46 revertants in TA98 and 247.5 revertants in TA100. The aqueous extracts were neither toxic nor mutagenic. However, the acid-treated aqueous extract was mutagenic to TA100. The organic material isolated from the acidic extract had an induced mutagenic potency of 44.2 revertants/mg extract. Normalizing these data indicate a mutagenic potency of 7.4 revertants/g MSWI ash leached.

Animals↗
PubMed1992

Mutation spectrum of spontaneous frameshift revertants in yeast using double-strand gap repair.

A mutation spectrum was constructed from a series of randomly isolated spontaneous His+ revertants of the frameshift mutant his4-38 in Saccharomyces cerevisiae. For each true revertant, a 438 bp region encompassing his4-38 on chromosome III was recovered into a shuttle vector by double-strand gap repair. Of the 45 independent His+ revertants sequenced, 44 were -1 base deletions and one revertant was a +2 base insertion. The -1 deletions exhibited a bimodal distribution. Of the bases encompassing the his4-38 region from +153-181, approximately 45% were not involved in a reversion event, although a -1 frameshift within this region will result in a viable His+ revertant. Approximately 49% of -1 events occurred within runs of 3 repeated bases. At these sites the strand-slippage model for frameshift mutation is supported. However, the -1 events occurring at sites of 2 repeated bases and the low frequency (2%) of +2 base insertions suggest that the transiently misaligned template model is a significant mechanism in reversion of his4-38. When the distribution of -1 events at repeated bases was discounted, a hotspot involving a -T at position +163 was resolved.

Base Sequence↗
PubMed1992

The relationship between nickel chloride-induced peroxidation and DNA strand breakage in rat liver.

Inorganic nickel chloride induces hepatic DNA strand breaks, chromosome aberrations, and lipid peroxidation under in vitro and in vivo conditions. The objective of this research was to determine if a relationship exists between NiCl2 genotoxicity and lipid peroxidation in vivo. Male Sprague-Dawley rats (210-250 g) were dosed with 0.56 or 0.75 mmol/kg NiCl2 subcutaneously and euthanized after specific time periods, ranging from 30 min to 24 hr. Livers were perfused and excised for the measurement of nickel content using atomic absorption spectrometry, lipid peroxidation using a thiobarbituric acid assay, and DNA strand breakage using single-stranded DNA extraction and the diaminobenzoic acid assay. The lower dose (0.56 mmol/kg) did not induce lipid peroxidation or strand breakage. The higher dose (0.75 mmol/kg) induced DNA strand breakage at 4 hr and lipid peroxidation at 12 hr in rat liver. Nickel was seen to accumulate in liver nuclei of rats receiving 0.75 mmol/kg. Deferoxamine (1 g/kg, ip, 15 min before the NiCl2 injection) completely inhibited DNA strand breakage at 4 hr but had no effect on lipid peroxidation. This suggests that lipid peroxidation is not causally related to genetic damage. NiCl2-induced DNA strand breakage may be caused by the induction of the Fenton reaction, generating hydroxyl radicals.

Animals↗
PubMed1991

Diethyldithiocarbamate suppresses the plant activation of aromatic amines into mutagens by inhibiting tobacco cell peroxidase.

Diethyldithiocarbamate is an antimutagen and repressed the activation of promutagens by plant systems. Earlier work implicated the involvement of tobacco cell (TX1) peroxidases in the plant cell activation of aromatic amines. We now present data that diethyldithiocarbamate represses the activation of 2-aminofluorene and m-phenylenediamine by inhibiting intracellular TX1 peroxidases under in vivo conditions. Concentrations of diethyldithiocarbamate that caused a 50% repression of TX1 cell activation of 2-aminofluorene and m-phenylenediamine also induced a 50% inhibition of TX1 cell peroxidase activity. Diethyldithiocarbamate in a concentration range between 25 and 500 microM directly inhibited peroxidase activity in TX1 cell homogenates in a concentration-dependent manner. Similar results were observed with purified horseradish peroxidase. The kinetics of peroxidase activity were studied in homogenates from control cells and cells treated with 750 microM and 25 mM diethyldithiocarbamate. There was no significant difference among the Km values among the three groups with a mean (+/- standard error) Km of 2.58 +/- 0.23 mM. However, the Vmax differed from 4.02 to 2.12 nmoles tetraguaiacol/min/micrograms protein, in the control and in the 25 mM diethyldithiocarbamate treatment group, respectively. These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.

Biotransformation↗
PubMed1990

The biochemical mechanisms of the plant activation of promutagenic aromatic amines.

Using specific monooxygenase and oxidase inhibitors in a plant cell/microbe coincubation assay, the biochemical mechanisms of the plant activation of two aromatic amines were compared. The biological endpoints included mutation induction, inhibition of mutagenicity, viability of the plant cells (activating system), and viability of the microbial cells (genetic indicator organism). The activation of m-phenylenediamine by TX1 cells was mediated by enzyme systems that were inhibited by diethyldithiocarbamate, potassium cyanide, methimazole, (+)-catechin or acetaminophen. The inhibition by metyrapone was attended by toxicity in the plant cells. These data implicate a TX1 cell peroxidase and a FAD-dependent monooxygenase in the plant activation of m-phenylenediamine. The TX1 cell activation of 2-aminofluorene was inhibited by diethyldithiocarbamate, 7,8-benzoflavone, acetaminophen or (+)-catechin. An additional pathway of the plant cells in the activation of 2-aminofluorene may involve a cytochrome P-448-type N-hydroxylase.

Acetaminophen↗
PubMed1990

Activation of promutagens by plant cell systems.

The plant cell/microbe coincubation assay is a sensitive and flexible assay for use in screening for plant-mediated promutagens as well as in basic mutation research. By using plant cell cultures of crop plants, xenobiotics used in agriculture can be evaluated for their mutagenic properties.

Biotransformation↗
PubMed1989

Use of the diaminobenzoic acid fluorescence assay in conjunction with uv absorbance as a means of quantifying and ascertaining the purity of a DNA preparation.

In the course of a study conducted to determine the correlation between covalently bound DNA-ethyl adducts and specific locus mutation induction in maize (W. E. Schy and M. J. Plewa Mutat. Res., 211, 231-241), it was necessary to accurately quantify and ascertain the purity of small amounts of DNA isolated from germinating maize kernels. DNA was purified from leaf primordial tissue that was dissected from germinating maize kernels and quantified by measuring its absorbance at 260 nm. Its absorbance at 260 nm relative to its absorbance at 280 nm (A260/A280 ratio) fell within the range of values that indicated a pure preparation. An attempt to verify the quantity of DNA using a second independent method specific for DNA, the diaminobenzoic acid dihydrochloride fluorescence assay, revealed a significant discrepancy between the two methods. The difference appeared to result from impurities present within the DNA preparation, despite a A260/A280 ratio that indicated otherwise. We found the A260/A280 ratio to be a poor indicator of the purity of DNA preparations, and determined that significant error may result from quantifying DNA using spectrophotometric methods alone. We propose as an alternative, quantifying DNA using the diaminobenzoic acid dihydrochloride assay in conjunction with uv absorbance at 260 nm and using a FLUOR/A260 ratio as an indicator of DNA purity.

Aminobenzoates↗
PubMed1989

Molecular dosimetry studies of forward mutation induced at the yg2 locus in maize by ethyl methanesulfonate.

The yg2 assay in Zea mays detects forward mutation in somatic cells within leaf primordia of embryos and it was used in an analysis of the molecular dosimetry of ethyl methanesulfonate (EMS). Parallel genetic and molecular dosimetry experiments were conducted in which the frequency of forward mutation and the level of covalently bound ethyl DNA adducts were determined. Prepared kernels were treated for 8 h at 20 degrees C with 1-10 mM EMS. EMS induced a direct concentration-dependent increase in mutation induction proportional to the exposure concentration (slope = 0.93). The kinetics of mutation induction demonstrated in the intact maize system were consistent with the kinetics observed earlier in in vitro model systems using cultured mammalian cells, and contrasted with the exponential increase in mutation induction characteristic of microbial species. Parallel molecular dosimetry experiments were conducted using [3H]EMS. DNA was extracted and purified from embryonic tissues containing the leaf primordia, the target tissue of the yg2 assay. A linear increase in the molecular dose was observed as a function of EMS concentration. Using concentration as a common parameter between the parallel genetic and dosimetry studies, mutation induction appeared to increase nearly in a direct proportion to the molecular dose. However, studies in other genetic systems indicate that the levels of specific DNA adducts, such as O6-ethylguanine (O6-EtGua) show a better correlation with mutation induction kinetics than molecular dose. Neither molecular dose, nor O6-EtGua levels account for differences in the absolute frequencies of mutation induction observed in different genetic systems. Therefore, reliable assessment of health risks posed to humans by chemical mutagens appears to require consideration of other factors in addition to DNA dose or adduct formation, including differences in repair capabilities and in the size of the genetic targets in humans relative to the model genetic systems under study.

Animals↗
PubMed1989

Effects of specific monooxygenase and oxidase inhibitors on the activation of 2-aminofluorene by plant cells.

Using specific inhibitors, a plant cell/microbe coincubation assay was employed to investigate biochemical mechanisms of plant activation. The biological endpoints of mutation induction, inhibition of mutagenicity and viability of the plant-activating system as well as viability of the microbiological indicator were simultaneously assayed from the same reaction tube. We investigated six inhibitors of monooxygenases and oxidases (diethyldithiocarbamate, methimazole, metyrapone, (+)-catechin, 7,8-benzoflavone and potassium cyanide). The activation of 2-aminofluorene by TX1 cells was mediated by an enzyme system(s) that was inhibited by microM amounts of diethyldithiocarbamate or 7,8-benzoflavone. (+)-Catechin (at low concentrations) or methimazole enhanced the activation of 2-aminofluorene while higher concentrations of (+)-catechin were inhibitory. These data indicate that a significant pathway of the plant activation of 2-aminofluorene is via a cytochrome P-448-type N-hydroxylase. The presence of a FAD-dependent monooxygenase was not detected.

Benzoflavones↗