Publications (2)8.48 Total impact
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Article: Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts
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ABSTRACT: Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5–20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or “Nature's pesticides” (the major toxic chemicals ingested by phytoplankton filterfeeders), and/or the result of human activities remains to be determined.Environmental and Molecular Mutagenesis 07/2006; 19(2):112 - 124. · 3.71 Impact Factor -
Article: In Vivo Transcription of nrdAB Operon and of grxA and fpg Genes Is Triggered in Escherichia coli Lacking both Thioredoxin and Glutaredoxin 1 or Thioredoxin and Glutathione, Respectively*
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ABSTRACT: We have previously described (1) that Escherichia coli maintains a balanced supply of deoxyribonucleotides by a regulatory mechanism that up-regulates the levels of ribonucleotide reductase with the lack of its main hydro-gen donors thioredoxin, glutaredoxin 1, and glutathione (GSH). By using a semi-quantitative reverse transcrip-tion/multiplex polymerase chain reaction fluorescent procedure that enables simultaneous analysis of up to seven mRNA species, we now demonstrate that regula-tion operates at the transcriptional level. Double mu-tant cells lacking both thioredoxin and glutaredoxin 1 had increased transcription of the nrdAB operon, as compared with the corresponding wild type parent (maximal induction of 10-and 9-fold for mRNA of nrdA and nrdB genes, respectively). Likewise, a dramatic in-crease of 36-fold in grxA mRNA was observed in bacteria simultaneously deficient in thioredoxin and GSH (the physiological reductant of all glutaredoxins). The in-creased expression of the grxA gene in trxA gshA double mutant bacteria was mimicked in trxA single mutant cells by depletion of GSH with diethylmaleate (DEM). This induction of grxA transcription was rapid since maximal increase was detected upon 10 min of DEM exposure. Like grxA expression, the basal level of fpg mRNA, encoding formamidopyrimidine-DNA glycosy-lase, was increased (about 4-fold) in a trxA gshA double mutant strain; this expression was also induced upon exposure to DEM (11-fold maximal induction). These re-sults suggest that transcription of grxA might share common redox regulatory mechanism(s) with that of the fpg gene, involved in the repair of 8-oxoguanine in DNA. Deoxyribonucleotides required for DNA synthesis are formed de novo by the enzyme ribonucleotide reductase (RRase) 1 (2). RRases, which catalyze the reduction of ribonucleotides to de-oxyribonucleotides, are divided into three main classes accord-ing to the mechanism employed to generate the free radical required for catalysis (3). Class I RRases are aerobic enzymes present in all higher organisms and in Escherichia coli. This bacterium actually contains the genetic information for two different class I RRases. One of them (called NrdAB and coded for by the nrdAB operon) is essential for growth in the presence of oxygen, whereas the other (NrdEF, encoded by the separate nrdEF operon) is normally not fully functional (4). No class II RRase has been found in E. coli, and class III enzymes operate only in anaerobiosis (5). The electrons for ribonucleotide reduction are supplied by thioredoxin (Trx) or glutaredoxin (Grx) in the case of class I RRase (6, 7). In the reduced form, both Trx and Grx contain two redox-active cysteine thiols, which by dithiol-disulfide inter-change reduce an acceptor disulfide in the active center of RRase. Reduced Trx is regenerated by thioredoxin reductase and NADPH, whereas oxidized Grx is reduced by two reduced glutathione (GSH) molecules with the formation of glutathione disulfide. The reduction of glutathione disulfide is catalyzed by glutathione reductase and NADPH. E. coli mutants defec-tive in Trx, Grx, or glutathione reductase and those defective in GSH biosynthesis have been named trxA, grx, gor, and gsh, respectively (8 –11). Apart from the first isolated glutaredoxin (Grx1 coded for by the grxA gene), E. coli contains two other glutaredoxins (called Grx2 and Grx3) (12). Like Grx1, both Grx2 and Grx3 show high activity as general GSH-disulfide oxidoreductases. Neverthe-less, Grx3 is an inefficient hydrogen donor for RRase in com-parison with Grx1 (about 5% of the catalytic activity of Grx1), whereas Grx2 lacks such activity (12). Recently, a glutare-doxin-like protein (called NrdH) with thioredoxin-like activity profile has been isolated from E. coli (13). NrdH is a functional hydrogen donor for RRase with higher specificity for the NrdEF than for the NrdAB enzyme. The physiological function of NrdH in E. coli is not well understood, since the nrdH gene is part of the poorly transcribed nrdEF operon (4). We have recently proposed that, apart from an assorted set of hydrogen donors and RRase activities, E. coli maintains a balanced supply of deoxyribonucleotides by a regulatory net-work that compensates the RRase, Trx, Grx1, and GSH levels (1). Of particular relevance is the large increase in ribonucle-otide reductase activity (from 19-to 23-fold) displayed by E. coli strains defective in both Trx and Grx1 (the two main hydrogen donors) (14) and the extremely high Grx1 content (55-fold) of * This work was subsidized by Grants PB95-0557-CO2-01 and PB95-0557-CO2-02 (DGES) and by Junta de Andalucía (groups CVI 0187 and CVI 0151). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of a fellowship from Junta de Andalucía (Spain).. 1 The abbreviations used are: RRase, ribonucleotide reductase; NrdAB, RRase coded for by the nrdAB operon; NrdEF, RRase coded for by the nrdEF operon; Trx, thioredoxin; Grx, glutaredoxin; GSH, re-duced glutathione; NrdH, protein coded for by the nrdH gene; RT/ MPCR, reverse transcription/multiplex polymerase chain reaction; PCR, polymerase chain reaction; DEM, diethylmaleate; GAPDH,Journal of Biological Chemistry 01/1998; 273:18382-18388. · 4.77 Impact Factor
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2006
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University of Cordoba (Spain)
- Departamento de Bioquímica y Biología Molecular
Córdoba, Andalusia, Spain
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