Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

The regulation of TORC1 pathway by the yeast chaperones Hsp31 is mediated by SFP1 and affects proteasomal activity.

The Saccharomyces cerevisiae heat shock proteins Hsp31-34 are members of DJ-1/ThiJ/Pfpl superfamily that includes human DJ-1 (Park7), a protein involved in heritable Parkinsonism. Although, homologs of these proteins can be found in most organisms their functions are unclear. We have carried out a quantitative proteomics analysis of yeast cells devoid of the whole set of Hsp31 family of proteins, as a model of Parkinson Disease (PD), under conditions of glucose availability and starvation. The protein profile indicates a constitutive activation of the enzyme TORC1 that makes the cells more sensitive to stress conditions. TORC1 activation prevents the cells from diauxic shift and entry into the stationary phase inducing cell death. Sfp1 stays at the helm among the several transcription factors governing the cell adaptation to Hsp31-34 deficiency. We show that Sfp1 remains mainly in the nucleus likely releasing TORC1 from inhibition by cytosolic Sfp1. Impairment of glycolysis leads to increased levels of methylglyoxal and accumulation of glycated proteins. We also show an increase in proteasome subunits in the Hsp31-34 mutant, under the control of Rpn4 transcription factor. This increase is abnormally accompanied by a decrease in proteasomal activity which could lead to accumulation of aberrant proteins and contributing to cell death.

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Thiol redox proteomics identifies differential targets of cytosolic and mitochondrial glutaredoxin-2 isoforms in Saccharomyces cerevisiae. Reversible S-glutathionylation of DHBP synthase (RIB3).

Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms.

[Prognostic factors associated with postoperative complications in liver transplant]. / Factores pronósticos de complicaciones postoperatorias en el transplante hepático.

OBJECTIVES: the postoperative evolution of patients submitted to orthotopic liver transplant (OLT) is frequently associated with the appearance of different types of complications such as renal failure, graft rejection, infections, and neurological disorders. These complications are the most significant causes of early morbidity and mortality in patients undergoing OLT. The purpose of the present study was the identification of factors related to the different postoperative complications after OLT. EXPERIMENTAL DESIGN: a prospective study was carried out. PATIENTS: seventy-eight variables were analyzed in 32 consecutive patients undergoing OLT. The factors independently associated with the appearance of postoperative complications were identified using a stepwise logistic regression analysis. RESULTS: the multivariate analysis showed that malondialdehyde and creatinine pretransplant serum levels were associated with the development of renal dysfunction. The pretransplant levels of haemoglobin and the units of platelets administered during surgery were prognostic factors of infections. Acute graft rejection was predicted by ?-glutamyl transpeptidase and total bilirubin serum levels. The pretransplant sodium and glutaredoxin levels in serum were associated with neurological complications. CONCLUSIONS: we propose these markers for the identification of high-risk patients allowing an early surveillance and/or treatment to improve morbidity and survival in patients submitted to OLT.

Factores pronósticos de complicaciones postoperatorias en el trasplante hepático / No disponible

Objetivo: la evolución postoperatoria de los pacientes sometidosa trasplante hepático ortotópico (THO) se encuentra frecuentementeasociada a la aparición de diversas complicaciones talescomo disfunción renal, rechazo agudo, infecciones y complicacionesneurológicas. Estas complicaciones constituyen las causasmás significativas de morbilidad y mortalidad tempranas en pacientesque reciben un THO. El propósito del presente estudio esla identificación de factores relacionados con las distintas complicacionespostoperatorias del THO. Diseño experimental: se llevóa cabo un estudio prospectivo.Pacientes: se analizaron 78 variables en 32 pacientes consecutivossometidos a THO. Utilizando un análisis de regresión logísticase identificaron aquellos factores asociados de forma independientecon la aparición de complicaciones postoperatorias.Resultados: el análisis multivariante demostró que los nivelespretrasplante en suero de malondialdehído y creatinina estabanasociados con el desarrollo de disfunción renal. Los niveles pretrasplantede hemoglobina y las unidades de plaquetas administradasdurante la cirugía fueron factores pronósticos de infecciones.El rechazo agudo fue pronosticado por los niveles séricos de γ-glutamiltranspeptidasa y de bilirrubina total. Los niveles pretrasplantede sodio y glutaredoxina en suero estuvieron asociados concomplicaciones neurológicas.Conclusiones: proponemos estos marcadores para la identificaciónde pacientes de alto riesgo, permitiendo una vigilanciay/o tratamiento anticipados que mejorarán la morbilidad y la supervivenciaen pacientes sometidos a THO

Objectives: the postoperative evolution of patients submittedto orthotopic liver transplant (OLT) is frequently associated withthe appearance of different types of complications such as renalfailure, graft rejection, infections, and neurological disorders.These complications are the most significant causes of early morbidityand mortality in patients undergoing OLT. The purpose ofthe present study was the identification of factors related to thedifferent postoperative complications after OLT. Experimental design:a prospective study was carried out.Patients: seventy-eight variables were analyzed in 32 consecutivepatients undergoing OLT. The factors independently associatedwith the appearance of postoperative complications wereidentified using a stepwise logistic regression analysis.Results: the multivariate analysis showed that malondialdehydeand creatinine pretransplant serum levels were associatedwith the development of renal dysfunction. The pretransplant levelsof haemoglobin and the units of platelets administered duringsurgery were prognostic factors of infections. Acute graft rejectionwas predicted by γ-glutamyl transpeptidase and total bilirubinserum levels. The pretransplant sodium and glutaredoxin levels inserum were associated with neurological complications.Conclusions: we propose these markers for the identificationof high-risk patients allowing an early surveillance and/or treatmentto improve morbidity and survival in patients submitted toOLT

Expression of glutaredoxin (thioltransferase) in the rat ovary during the oestrous cycle and postnatal development.

Glutaredoxins (Grxs) are low-molecular-weight proteins which participate in redox events in association with glutathione (GSH) and are involved in a variety of cellular processes. It is known that oxidative stress plays important physiological roles within the ovary. In the present study, we have prepared specific antibodies against rat Grx and have used them to localize the protein in the ovaries of rats during postnatal development and during the oestrous cycle, by immunohistochemical methods. We have also performed a quantitative analysis of Grx by ELISA and Western blotting in homogenates of whole ovaries of cycling and pseudopregnant rats. We have found a prominent presence of Grx in the oocytes and in corpora lutea (CL) during developmental and oestrous cycle changes. Grx was absent from the oocytes in the first days of postnatal life when marked oocyte degeneration takes place, but its presence was very conspicuous in the cytoplasm of oocytes in healthy and attretic follicles in rats from 10 days of age onward, independently of the day of oestrous cycle. Follicular cells were negative. Grx immunostaining in the CL was strong in infiltrating macrophages and in a population of steroidogenic cells that survived the apoptotic burst in regressing CL and in CL remnants, but was faint or absent in young CL of the current cycle and in CL during pseudopregnancy. Grx content and oxidoreductase activity in whole ovaries increased significantly during the phase transition from proestrous to oestrous along the cycle. These results support a role of Grx in the maintenance of functional oocytes and in luteal cells surviving the regression process, probably as a consequence of the demonstrated deglutathionylating function of this protein in an antioxidant and antiapoptotic context.

Immunolocalization of glutaredoxin in the human corpus luteum.

Glutaredoxin (Grx) is a small protein with oxidoreductase activity which is involved in the cellular defence against oxidative stress. Corpus luteum (CL) regression has been related to the generation of reactive oxygen species (ROS). We have studied the presence of glutaredoxin in the human ovary during the ovulatory cycle using polyclonal antibodies developed against recombinant human Grx. Immunostaining was only detected between days 15 and 23 of the cycle and was localized exclusively in the corpus luteum. Grx-positive cells corresponded to granulosa-derived luteal cells (GLC) whereas the remaining luteal cell types were not immunostained. In general, Grx immunoreactivity was parallel to the functional activity of the CL. Most GLC were immunostained on days 15-16 of the cycle, whereas on days 17-19 immunoreaction was found mainly at the inner and outer aspects of the granulosa lutein layer (GLL). After this stage only isolated GLC showed Grx immunoreactivity and no reaction was found from day 23 of the cycle onward. In two CL of pregnancy that were also studied, isolated GLC showed Grx immunoreactivity. Loss of Grx immunoreactivity was coincident with the appearance of morphological signs of structural luteolysis, such as shrinkage of the GLL and the presence of apoptotic cells. These data suggest that Grx, as a cellular antioxidant, plays an important role in the mechanisms of human CL development.

Redox regulation of c-Jun DNA binding by reversible S-glutathiolation.

Redox control of the transcription factor c-Jun maps to a single cysteine in its DNA binding domain. However, the nature of the oxidized state of this cysteine and, thus, the potential molecular mechanisms accounting for the redox regulation of c-Jun DNA binding remain unclear. To address this issue, we have analyzed the purified recombinant c-Jun DNA binding domain for redox-dependent thiol modifications and concomitant changes in DNA binding activity. We show that changes in the ratio of reduced to oxidized glutathione provide the potential to oxidize c-Jun sulfhydryls by mechanisms that include both protein disulfide formation and S-glutathiolation. We provide evidence that S-glutathiolation, which is specifically targeted to the cysteine residue located in the DNA binding site of the protein, may account for the reversible redox regulation of c-Jun DNA binding. Furthermore, based on a molecular model of the S-glutathiolated protein, we discuss the structural elements facilitating S-glutathiolation and how this modification interferes with DNA binding. Given the structural similarities between the positively charged cysteine-containing DNA binding motif of c-Jun and the DNA binding site of related oxidant-sensitive transcriptional activators, the unprecedented phenomenon of redox-triggered S-thiolation of a transcription factor described in this report suggests a novel role for protein thiolation in the redox control of transcription.

Purification and characterization of multiple glutathione transferase isoenzymes from grey mullet liver.

Fourteen isoforms of glutathione S-transferase (GST) have been separated and purified from mullet (Mugil cephalus) liver by scaling up an automatic analytical method based on anionic exchange chromatography. The activity of each isoenzyme with several substrates was determined. Dimeric combinations of six subunits make up this heterogeneous isoenzyme population. Five of these were resolved by reverse phase chromatography; four of them, named a, b, c and d, were present in more than one isoform, had the same apparent molecular mass (25.2 kDa) by SDS-PAGE, and were immunochemically related to plaice GST-A and possibly to rat GST-5 but not to plaice GST-B or any other rat GST subunit; they would belong to the theta class. Subunit e was only present in isoenzyme I which was basic, had an apparent molecular mass of 23.4 kDa and would belong to the alpha class, since it was recognized by antibodies towards plaice GST-B and rat GST-1 and GST-8 and less intensely by anti-(rat)GST-2. Another subunit, named f, with 25.2 kDa apparent molecular mass that could not be distinguished by reverse phase chromatography, was detected immunochemically by positive reaction with antibodies to rat GST-1 and GST-2 in addition to reaction with anti-(plaice)GST-A. As suggested by these results we discuss the existence of genetic polymorphism, the differential expression and the evolutionary relationships of mullet GSTs.

Changes in GST-isoenzyme pattern of some organs of sheep exposed to different levels of pollution.

GST isonzyme patterns were studied in the cytosolic fraction of liver, kidney and lung of sheep exposed to industrial metal pollutants and compared with those of control animals. The methodology included the determination of enzymatic activities with several subunit-specific substrates (DCNB, NPB, EPNP and EA) and Western blotting using antibodies to specific rat GST subunits 1, 8 (alpha class), 3 (mu class) and 7 (pi class). In liver and lung, crossed reactivities with subunits 1 and 3 were absent in the controls but were present in exposed animals. Just the opposite result was obtained for subunit 8 crossed reactivity that was only in the control animals. In the kidney, crossed reactivities towards subunits 3 and 8 were absent and crossed reactivity equivalent to subunit 7 was present in all animals, and equivalent to subunit 1 was weakly induced in exposed animals. A 3.3-fold increase in the activity with NPB detected in the kidneys of exposed animals points to the induction of a theta class isoenzymes. Clear increases were found in the livers of exposed animals in the activities with CDNB (1.8-fold), DCNB (2.6-fold) and EPNP (2.1-fold), but no differences were found in the lungs with any of the substrates. The GST isoenzyme pattern of liver and lung could be, in principle, a useful biomarker of exposure to environmental pollution in sheep.

Characterization of mammalian thioredoxin reductase, thioredoxin and glutaredoxin by immunochemical methods.

Specific polyclonal antibodies towards the oxidized form of bovine thioredoxin reductase (TR) have been obtained in rabbits, and purified. The antigenicity was lost upon reduction of TR by NADPH indicating a large conformational change upon reduction of the redox-active disulfide in the enzyme. The antibodies did not cross-react with other bovine NADPH-dependent dehydrogenases. No reactivity was observed with TR from bacteria, yeast or rat and only a slight reaction was obtained with TR from horse. Immunoaffinity purified anti-thioredoxin and anti-glutaredoxin antibodies were used to develop competitive indirect ELISA assays that were validated giving very good linearity, reproducibility, sensitivity and parallelism. The glutaredoxin (Grx) immunoassay is the first quantitative method described to measure the protein. When applied to a battery of calf tissues the contents of Grx varied from 7 to 120 micrograms per gram of fresh tissue. Skeletal and heart muscles gave the lowest values and spleen and salivary glands the highest. However, skeletal muscle showed the highest gluthathione-hydroxyethyl disulfide oxidoreductase specific activity.

Purification from placenta, amino acid sequence, structure comparisons and cDNA cloning of human glutaredoxin.

Glutaredoxin is generally a glutathione-dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)-disulfide-oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH-dependent hydrogen-donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three-dimensional structure in solution to identify conserved residues and predict a structure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.

Immunochemical characterization and tissue distribution of glutaredoxin (thioltransferase) from calf.

Glutaredoxin catalyzes glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH and glutathione reductase and has an active site disulfide/dithiol with the sequence -Cys-Pro-Tyr-Cys-. Calf thymus glutaredoxin (thioltransferase), which contains two additional structural half-cystine residues, was purified to homogeneity, using a modification of the previously described isolation procedure. This method involved a pI-shift of glutaredoxin, obtained after oxidation of the fully reduced form with hydroxyethyl-disulfide, followed by CM-Sepharose chromatography. On both SDS- and IEF-gels the protein migrated as one band (M(r) 12,000). The pure protein was used to affinity-purify rabbit antiglutaredoxin antibodies obtained by immunization with the oxidized form of glutaredoxin. Using these antibodies the distribution of glutaredoxin was mapped in calf organs and tissues by Western blots and by immunohistochemistry. Glutaredoxin was demonstrated in all organs investigated. Western blots showed the presence of weak additional high molecular weight bands of unknown identity in certain organs. The immunohistochemical analyses revealed that glutaredoxin is highly expressed in a wide variety of cell types, both epithelial and mesenchymal. The distribution and occurrence in the calf organs was similar to that previously described for thioredoxin in the rat. There were some exceptions: e.g., follicular cells in the ovary did not contain immunohistochemically demonstrable glutaredoxin but expressed thioredoxin. Particularly striking were observations of strong glutaredoxin immunoreactivity in oocytes in the ovary and the pattern of glutaredoxin in epithelial tissue of the skin and tongue reflecting differential expression during cell differentiation. The distribution demonstrated that glutaredoxin serves functions apart from the originally described role as hydrogen donor for ribonucleotide reductase which only occurs in replicating cells. Such functions should relate particularly to glutathione-catalyzed protein disulfide oxidoreductions and cellular signalling by redox regulating mechanisms.

Topological relationships between porcine anterior pituitary hormones and the thioredoxin and glutaredoxin systems.

Thioredoxin (TRX) and glutaredoxin (GRX) had previously been localized in folliculo-stellatae (FS) cells and in only a fraction of glandular cells of the anterior pituitary (Padilla et al., 1992). Here we report on a double immunolabelling study carried out to determine the correlation between the type of secretory cell and the presence of TRX or GRX. TRX and GRX levels were under the detection limits in gonadotropes, thyrotropes and corticotropes. A considerable proportion of lactotropes contained TRX or GRX; a higher proportion of somatotropes contained TRX and all of them were GRX-positive. The secretory cell types more frequently detected in the epithelium of well-defined follicles lined by TRX-positive FS cells were somatotropes and lactotropes followed by corticotropes; gonadotropes and thyrotropes were scarce in these structures. Regarding the biological functions of glutaredoxin and thioredoxin, these results show that involvement in the processing of secretory proteins is not a general property of these two thiol-disulfide oxidoreductases, not even specifically in the case of cysteine-rich secretory proteins. On the other hand, another type of functional specificity perhaps related to the heterogeneous response of the endocrine cells is discussed.

Regulation of horse-liver glutathione reductase.

1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations. 2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective. 3. GSSG fully reactivated the inactive enzyme at 38 degrees C and neutral to acidic pH (5.5-7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

Horse-liver glutathione reductase: purification and characterization.

1. Purification of horse-liver glutathione reductase was obtained by affinity chromatography on N6-(6-aminohexyl)-adenosine-1'5'-bisphosphate Sepharose (N6-2'5'-ADP-Sepharose) and Reactive Red-120-Agarose, and chromatography on DEAE-Sephadex and Sephacryl S-300. 2. The final preparation had 248 U/mg specific activity after 11,174-fold purification with 47% final recovery, and was homogeneous by SDS-electrophoresis. It showed charge heterogeneity in non-denaturing electrophoresis and chromatofocusing, with several peaks of pI between 5.7 and 6.7. 3. The enzyme was homodimeric (107,000 native MW), with S20w = 6.31 S, and 41.22 A of hydrodynamic radius. It showed absorption peaks at 270, 370 and 462 nm, a characteristic of flavoproteins. 4. When NADPH was substituted by deamino-NADPH or NADH the enzyme showed 69 and 8.5% activity, respectively, while with glutathione-CoA mixed disulfide the enzyme had 23% of the activity shown with GSSG. Apparent Km values of 8.8, 680, 59, and 560 microM were measured for NADPH, NADH, GSSG and ferricyanide, respectively.

Biochemical and genetic indices of marine pollution in Spanish littoral.

Increased activities of several detoxifying and antioxidant enzymes were detected in mollusc and fish from Spanish littoral areas with high metal contents. Ethanolic extracts from molluscs contained direct-acting and polar genotoxins of oxidative type, which were detected by strain TA102 of S. typhimurium and catalase-deficient strains of E. coli. Animals from contaminated sites contained less genotoxins than those from control areas. Polluted fishes displayed highly induced cytochrome P-450 activity and increased promutagen activation capabilities. In addition, specific forms of glutathione transferase and superoxide dismutase were induced, particularly highly acidic forms.