A signature of circulating microRNAs predicts the response to treatment with FOLFIRI plus aflibercept in metastatic colorectal cancer patients.

The benefit of adding the antiangiogenic drug aflibercept to FOLFIRI regime in metastatic colorectal cancer (CRC) patients resistant to or progressive on an oxaliplatin-based therapy has been previously demonstrated. However, the absence of validated biomarkers to predict greater outcomes is a major challenge encountered when using antiangiogenic therapies. In this study we investigated profiles of circulating microRNAs (miRNAs) to build predictive models of response to treatment and survival. Plasma was obtained from 98 metastatic CRC patients enrolled in a clinical phase II trial before receiving FOLFIRI plus aflibercept treatment, and the circulating levels of 754 individual miRNAs were quantified using real-time PCR. A distinct signature of circulating miRNAs differentiated responder from non-responder patients. Remarkably, most of these miRNAs were found to target genes that are involved in angiogenic processes. Accordingly, some of these miRNAs had predictive value and entered in predictive models of response to therapy, progression of disease, and survival of patients treated with FOLFIRI plus aflibercept. Among these miRNAs, circulating levels of hsa-miR-33b-5p efficiently discriminated between responder and non-responder patients and predicted the risk of disease progression. Moreover, the combination of circulating VEGF-A and miR-33b-5p levels improved clinical stratification of metastatic CRC patients who were to receive FOLFIRI plus aflibercept treatment. In conclusion, our study supports circulating miRNAs as valuable biomarkers for predicting better outcomes in metastatic CRC patients treated with FOLFIRI plus aflibercept.

Diagnostic potential of NETosis-derived products for disease activity, atherosclerosis and therapeutic effectiveness in Rheumatoid Arthritis patients.

OBJECTIVES: 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). METHODS: One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. RESULTS: NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. CONCLUSIONS: NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

'Atherothrombosis-associated microRNAs in Antiphospholipid syndrome and Systemic Lupus Erythematosus patients'.

MicroRNAs markedly affect the immune system, and have a relevant role in CVD and autoimmune diseases. Yet, no study has analyzed their involvement in atherothrombosis related to APS and SLE patients. This study intended to: 1) identify and characterize microRNAs linked to CVD in APS and SLE; 2) assess the effects of specific autoantibodies. Six microRNAs, involved in atherothrombosis development, were quantified in purified leukocytes from 23 APS and 64 SLE patients, and 56 healthy donors. Levels of microRNAs in neutrophils were lower in APS and SLE than in healthy donors. Gene and protein expression of miRNA biogenesis-related molecules were also reduced. Accordingly, more than 75% of identified miRNAs by miRNA profiling were underexpressed. In monocytes, miR124a and -125a were low, while miR-146a and miR-155 appeared elevated. Altered microRNAs' expression was linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in both pathologies. In vitro treatment of neutrophils, monocytes, and ECs with aPL-IgG or anti-dsDNA-IgG antibodies deregulated microRNAs expression, and decreased miRNA biogenesis-related proteins. Monocyte transfections with pre-miR-124a and/or -125a caused reduction in atherothrombosis-related target molecules. In conclusion, microRNA biogenesis, significantly altered in neutrophils of APS and SLE patients, is associated to their atherothrombotic status, further modulated by specific autoantibodies.

Desarrollo y validación de un protocolo para el análisis proteómico de muestras de broncoaspirado de pacientes con adenocarcinoma de pulmón / Development and validation of a protocol for the proteomic analysis of bronchoaspiration samples in patients with lung adenocarcinoma

INTRODUCCIÓN: la identificación de marcadores en el broncoaspirado puede representar un hallazgo relevante, complementario al estudio citohistológico. OBJETIVO: determinar si el procedimiento aplicado es válido para obtener péptidos y proteínas suficientes, normalizar el protocolo y realizar un análisis shotgun por espectrometría de masas de los péptidos resultantes. MATERIAL Y MÉTODOS: se incluyeron 4 muestras de broncoaspirado de pacientes con adenocarcinoma de pulmón, aplicando el siguiente flujo de trabajo (I) recolección del broncoaspirado; (II) purificación de su componente proteica; (III) digestión proteica en solución; y (IV) análisis shotgun por espectrometría de masas de los péptidos resultantes. Para clasificar las proteínas según componente celular, proceso biológico y función molecular, se realizó un análisis Gene Onthology. RESULTADOS: en la muestra de 2,1 ml de broncoaspirado medio por paciente, fijando una tasa de falso descubrimiento (FDR, False Discovery Rate) restrictiva del 1%, se identificaron un número elevado de proteínas, con una media de 625, rango entre 529 a 708, y 6.290 péptidos únicos, rango entre 5.585 a 6.759. La composición proteica de las muestras fue homogénea, sin diferencias en la clasificación en relación a los componentes celulares, procesos biológicos y funciones moleculares. La eficacia de la digestión enzimática fue mayor del 90% en todos los casos, lo que asegura la reproducibilidad de la metodología utilizada. CONCLUSIONES: la metodología desarrollada para el análisis proteómico es altamente eficaz y reproducible, identificando un número elevado de proteínas en el broncoaspirado y permitirá realizar, de manera robusta, un estudio cuantitativo en una muestra amplia de pacientes y grupo control

INTRODUCTION: identifying markers in aspirated bronchial samples could represent a relevant discovery, complementary to cytohistological studies, in patients with lung adenocarcinoma. OBJECTIVE: determine whether the procedure applied is valid to obtain sufficient peptides and proteins to establish a protocol and perform mass spectrometry-based shotgun proteomic analysis for the resulting peptides. Material and METHOD: 4 aspirated bronchial samples were included from patients with pulmonary adenocarcinoma, applying the following work flow (I) recollection of aspirated bronchial samples; (II) purification of its protein component; (III) protein digestion in solution; and (IV) mass spectrometry-based shotgun proteomic analysis for the resulting peptides. To classify proteins based on cellular component, biological process and molecular function, gene ontology analysis was performed. RESULTS: in the 2.1 mlmeanbronchial sample per patient, a 1% restrictive false discovery rate (FDR)was established;an elevated number of proteins were identified, with an average of 625, range between 529 to 708 and 6.290 single peptides, range between 5.585 to 6.759. The proteincomposition of the samples was uniform, without differences in the classification with regards to cellular components, biological processes and molecular functions. The efficacy of enzymatic digestion was greater than 90% in all cases, which guarantees the reproducibility of the methodology used. CONCLUSIONS: themethodology developed for the proteomic analysis is highly effective and reproducible; it identifies a high number of proteins in the bronchial samples and allows us to perform, in a robust manner, a quantitative study in a wide range of patients and control group

Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction.

UNLABELLED: Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. SIGNIFICANCE: Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases.

Nitric oxide and cancer: the emerging role of S-nitrosylation.

Nitric oxide (NO˙) is a short-lived, endogenously produced gas that is highly diffusible across cell membranes and acts as a signaling molecule in the body. The redox state and chemistry of NO˙ facilitate its interaction with various proteins thus regulating various intracellular and intercellular events. One of the key mechanisms by which NO˙ regulates the function of various target proteins is through the coupling of a nitroso moiety from NO-derived metabolites to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process commonly known as S-nitrosylation. S-nitrosylation signaling events within the cell have led to the discovery of many other physiological functions of NO˙ in many other types of cells including cancer cells. Only recently are the diverse roles of S-nitrosylation in cancer beginning to be understood. In the present review we discuss the recent evidence for the diverse roles of NO˙/SNO-related mechanisms in cancer biology and therapy, including the participation of NO˙ in the pathogenesis of cancer, its duality in protecting against or inducing cancer cell death and the contribution of NO˙ to metastatic processes. In addition, NO˙ can be therapeutically used in the reversal of tumor cell resistance to cytotoxic drugs and as a sensitizing agent to chemo- and radiotherapy. Finally, recent studies providing evidence for NO-related mechanisms of epigenetic gene expression regulation will also be discussed. Undoubtedly, new exciting results will contribute to this rapidly expanding area of cancer research.

[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

Mecanismos de lesión hepatocelular / Mechanisms of liver cell injury

No disponible

Treatment of refractory cholestatic pruritus with molecular adsorbent recirculating system (MARS).

UNLABELLED: Pruritus is a common complication of cholestatic liver diseases or liver graft dysfunction. Current medical therapies lack efficacy. The molecular adsorbent recirculating system (MARS) represents an interesting therapeutic option. Our objective was to report our experience in the management of four patients with intractable pruritus with MARS. PATIENTS AND METHODS: The MARS treatment cycle included three consecutive treatments, each of 8 hours duration. The four patients with intractable pruritus who were treated had primary biliary cirrhosis/autoimmune hepatitis overlap syndrome (n = 1), ductopenic allograft rejection (n = 2), or posttransplant cholestatic HCV recurrence (n = 1). Intensity of pruritus was documented 24 hours before as well as 24 hours, 7 and 30 days after MARS therapy, and at the end of follow-up. We measured complete blood cell counts, glucose, BUN, creatinine, sodium, potassium, AST, ALT, GGT, alkaline phosphatase, bilirubin, prothrombin activity, and activated partial thromboplastin time. RESULTS: MARS therapy was well tolerated. Patient 1 experienced temporal relief of pruritus, but needed another MARS cycle because of relapse. Patient 2 experienced partial and temporary relief of pruritus, was listed for retransplantation, and received a liver graft 2 months later. Patient 3 showed a dramatic reduction in the degree of pruritus with MARS. Pruritus in patient 4 decreased promptly with MARS therapy and conversion of immunosuppression to tacrolimus, thereby avoiding retransplantation. CONCLUSION: MARS therapy is a promising, safe therapeutic option to treat refractory pruritus caused by cholestatic liver disorders.

Uptake and clearance of PCB congeners in Chamaelea gallina: response of oxidative stress biomarkers.

PCB uptake and clearance by clams, Chamaelea gallina, were studied in specially designed flow-through channels. After 8 weeks exposure to 10 ppb Aroclor 1254 in water, clams were depurated for 10 weeks, in the same exposure channel or after transfer to clean systems. Accumulation of the 20 congeners studied depended on its initial abundance and physicochemical properties. A linear relationship was found between log bioconcentration factor and log octanol/water partition coefficient of each form. Clearance of each PCB depended also on its initial load and solubility, being faster in clams transferred to clean systems. Exposure significantly enhanced catalase and 6-P-gluconate dehydrogenase activities, but not other antioxidative enzymes. Superoxide dismutase, low during the exposure phase, increased seven-fold during depuration. Aroclor-treated clams had higher GSH levels than controls, but decreased to 15-35% after 2 days clearance, rose to 150% after 12 days, and declined to low levels by the end of the experience. Biotransformation of PCBs to quinones and redox cycling-promoted oxidative stress might explain the increased antioxidative defenses. The biochemical changes observed at the beginning of clearance could be attributed to clam handling, by adaptation to and recovery from hypoxic/anoxic stress.

Rapid induction of NF-kappaB binding during liver cell isolation and culture: inhibition by L-NAME indicates a role for nitric oxide synthase.

This study is the first to demonstrate activation of NF-kappaB binding just 10 minutes into the commonly employed hepatocyte isolation procedure. It is further reported that the anti-oxidant Trolox can prevent the induction of NF-kappaB during the well established hepatocyte isolation procedure but not during their subsequent culture. However both phases of NF-kappaB activation are inhibited by L-NAME intimating a role for NO production, via nitric oxide synthase. These findings demonstrate that at least 2 different signal transduction pathways are operative during hepatocyte isolation and culture. Thus further studies employing Trolox and L-NAME will help delineate how each pathway contributes to the generalised loss of liver function commonly observed in vitro.

Content of 8-oxodG in chromosomal DNA of Sparus aurata fish as biomarker of oxidative stress and environmental pollution.

The 8-oxodG content has been measured in chromosomal DNA of gilthead seabream (Sparus aurata) by HPLC-EC. Susceptibility of different tissues to oxidative DNA damage was studied by exposing fish to model pollutants. Cu(II), paraquat (PQ) and malathion failed to promote DNA oxidation in liver, while dieldrin significantly increased the 8-oxodG content in this organ, but not in gills or blood. After PQ exposure, fish liver showed high levels of glucose-6-P dehydrogenase (G-6PDH) and GSSG reductase activities. The increased antioxidant status and the lack of a specific transport system could explain the lack of susceptibility of liver to DNA oxidative damage induced by PQ. Increased levels of 8-oxodG were detected in the gills of PQ-exposed fish after 8 and 24 h. In contrast, after 48 h exposed fish contained lower 8-oxodG levels than controls. The existence of a PQ transport system in this O2-rich organ and the lack of a significant increase in antioxidant defenses would explain the sensitivity of gills to DNA damage promoted by PQ. Elimination of this soluble chemical and the putative induction of DNA-repair enzymes specific for oxidative damages could explain the drop of 8-oxodG levels at longer times. Fish exposed to moderate levels of urban and industrial pollution showed significantly high 8-oxodG content in hepatic DNA. We conclude that 8-oxodG determination in chromosomal DNA by HPLC-EC is a potentially useful biomarker of environmental pollution, although its response is still somewhat lower than that of other well-established biomarkers of oxidative stress.

Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences.

This paper examines the relationship in Escherichia coli between the in vivo content of 8-oxoguanine (8-oxoG) in chromosomal DNA and deficiencies of various key antioxidant defences. The structural genes for catalases (katG and katE), cytosolic superoxide dismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase (fpg) were inactivated to obtain bacterial strains lacking the scavenger enzymes for H2O2 or O2.- or the DNA repair protein for 8-oxoG. Wild-type bacteria showed 5-fold increased sensitivity to both lethality and mutagenesis by H2O2 in K medium (1% casamino acids and 1% glucose), as compared with nutrient broth. This higher sensitivity was associated with increased chromosomal oxidative damage, estimated as the 8-oxodG content, and with a marked decrease in both catalase and SOD activities. Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayed increased 8-oxodG content in chromosomal DNA (2.8-fold that of the wild-type) when grown under standard aerated conditions. Comparatively, no significant difference in 8-oxodG content was observed in cells grown without aeration. Bacteria totally devoid of catalase activity (katG katE mutant) showed wild-type contents of 8-oxodG in chromosomal DNA when grown under aerated conditions. Nevertheless, the protective role of catalase in preventing formation of 8-oxodG in chromosomal DNA became evident under oxidative stress conditions: growth under hyperoxygenation and, particularly, following H2O2 exposure. Catalase deficiency resulted in a dramatic decrease in viability after H2O2 exposure. A deficiency of Fpg protein also sensitized E.coli to H2O2 lethality, though to lesser extent than a deficiency of catalase activity. However, the scavenger enzyme and the DNA repair protein protected equally against 8-oxoG formed in vivo upon H2O2 treatment.

Mutagenic activation of aromatic amines by molluscs as a biomarker of marine pollution.

Mutagenic activation of arylamines by mollusc S9 fractions was evaluated as a biomarker for marine pollution. Two bivalve species were used as bioindicators, the common mussel (Mytilus edulis) and the striped venus (Chameleo gallina). A strain of Salmonella typhimurium overproducing O-acetyltransferase was used as indicator of mutagenicity. Mussels from an area of the North Atlantic Spanish zone that was exposed to an accidental crude oil spill were compared to bivalves from a reference area. C. gallina samples were from low polluted and highly polluted areas of the South Atlantic Spanish littoral. The promutagen 2-aminoanthracene (2-AA) was activated to mutagenic derivative(s) by S9 fractions from both C. gallina and M. edulis. Animals from contaminated sites showed higher arylamine activation capabilities than reference animals. This was further correlated with the mutagenic activities of corresponding cyclopentone-dichloromethane animal extracts. 2-AA activation by mollusc S9 was potentiated by alpha-naphthoflavone (ANF), known to inhibit PAH-inducible CYP1A cytochromes from vertebrates, but inhibited by methimazole (MZ), a substrate of the flavin monooxygenase (FMO) system. 2-AA-activating enzymes were mainly cytosolic; this localization clearly suggests that such activity could be attributed to soluble enzymes, different from the CYP1A or FMO systems. In conclusion, mutagenic activation of arylamines by mollusc S9, using as indicator a strain of Salmonella typhimurium that overproduces O-acetyltransferase, could be a reliable biomarker for marine pollution.

The levels of ribonucleotide reductase, thioredoxin, glutaredoxin 1, and GSH are balanced in Escherichia coli K12.

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.

Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants.

Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism. To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used. Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats. The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system. This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics. The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic. The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity. The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.

Rapid determination of glutathione status in fish liver using high-performance liquid chromatography and electrochemical detection.

A rapid and sensitive method for the detection of reduced (GSH), oxidised (GSSG) and protein-bound (PSSG) glutathione in fish liver, using reversed-phase HPLC with electrochemical detection has been developed. Separation was carried out isocratically at room temperature using 0.020 M sodium phosphate, pH 2.7 as mobile phase. A series dual-channel electrochemical detector was used for the simultaneous determination of GSH and GSSG. PSSG was determined after reduction by 1,4-dithiothreitol. The detection limits found for a 3:1 signal-to-noise ratio were 16.2 and 8.1 pmol for GSH and GSSG, respectively. The results obtained demonstrate that this method could be useful for measurement of the glutathione redox status in fish liver and are consistent with those reported for other fish. The method has been applied to follow the oxidative stress induced in vivo by copper(II) ions in the gilthead seabream fish (Sparus aurata). At longer times after copper(II) injection, the glutathione redox status of the exposed fish returned to a more reduced state, suggesting the existence of adaptive processes.

Promutagen activation by fish liver as a biomarker of littoral pollution.

Metabolic activation of known promutagens by liver S9 fractions of Mugil sp. (grey mullet) from two zones of the South Atlantic Spanish littoral was determined and related to their pollution levels. Sediments from the putative contaminated area contained high concentrations of PAHs, PCBs and pesticides, and animals from the polluted site exhibited higher concentrations of metals than those from the reference area. Hepatic S9 fractions of mullets from the polluted site showed 5.1-, 18.6- and 42.8-fold higher capability to activate benzo(a)pyrene, 2-acetyl-aminofluorene and 2-aminoanthracene, respectively, than those from reference animals. Cadmium, a highly toxic metal, was one of the pollutants detected in the contaminated area. Gilthead seabream (Sparus aurata) were exposed under controlled conditions to different Cd concentrations in order to investigate the effects of Cd on fish promutagen activation capability. A clear dose-response relationship was observed between Cd concentration, EROD activity and metabolic activation of 2-aminoanthracene and benzo(a)pyrene. Our data indicate that the enhanced promutagen activation by fish S9 fractions accompanying induction of EROD activity is a sensitive and reliable index of pollution in aquatic environments.