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The evaluation of antioxidant system capacity is important in aquatic toxicology. It was aimed to characterize the liver antioxidant enzymes (SOD, CAT, GPX, GR, and GST) and to test the in vitro Zn effect (200 and 400 ZnSO 4 μg/L) in native fish Barbus meridionalis obtained from the Osor River (NE, Spain) influenced by Zn contamination. The maximal enzyme activities were at pH 7.0-7.5 and 100 mM phosphate buffer. Barbel showed high catalytic activity (high Vmax and low Km) indicating the efficient antioxidant detoxification ability. Direct Zn effect caused an antioxidant system imbalance. Mostly upon lower Zn concentration, GPX activity decreased (95-100 %) though CAT, GR, and GST increased (36-1543 %). GSH values either stimulated (290 %) or inhibited (85-93 %) due to tissue differences. The first record of barbel antioxidant enzyme characterization and in vitro data presenting an unbalanced antioxidant pattern could be significant to evaluate the metal pollution in the Osor River for further in vivo studies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Pediatric hepatic steatosis is highly prevalent and closely related to type 2 diabetes. This study aimed to determine whether the addition of supervised exercise to a family-based lifestyle and psycho-educational intervention results in greater reduction of percentage of hepatic fat (HF), adiposity, and cardiometabolic risk factors in children with overweight/obesity., Research Design and Methods: The study subjects of this nonrandomized, two-arm, parallel design clinical trial were 116 overweight/obese children (10.6 ± 1.1 years of age, 53.4% girls) living in Vitoria-Gasteiz (Spain). For 22 weeks, they followed either a lifestyle and psycho-education program (control intervention [CInt], N = 57), consisting of two family-based education sessions/month, or the same plus supervised exercise (intensive intervention [II], N = 59) focused mainly on high-intensity aerobic workouts (3 sessions/week, 90 min/session). The primary outcome was the change in percentage of HF (as measured by MRI) between baseline and the end of the intervention period. Secondary outcomes included changes in BMI, fat mass index (FMI), abdominal fat (measured by DEXA), blood pressure, triglycerides, HDL, LDL, γ-glutamyl transferase, glucose, and insulin concentrations., Results: A total of 102 children completed the trial ( N = 53 and N = 49 in the CInt and II groups, respectively). Percentage of HF decreased only in the II group (-1.20 ± 0.31% vs. 0.04 ± 0.30%, II and CInt groups, respectively), regardless of baseline value and any change in adiposity ( P < 0.01). BMI, FMI, abdominal fat ( P ≤ 0.001), and insulin ( P < 0.05) were reduced in both groups., Conclusions: Multicomponent intervention programs that include exercise training may help to reduce adiposity, insulin resistance, and hepatic steatosis in overweight/obese children., (© 2019 by the American Diabetes Association.)

Background and Objective: Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a complex multisystemic severe drug hypersensitivity reaction whose diagnosis and management are troublesome. DRESS syndrome requires management by various specialists. The correct identification of the culprit drug is essential to ensure safe future therapeutic options for the patient. There are no previous Spanish guidelines or consensus statements on DRESS syndrome. Objective: To draft a review and guidelines on the clinical diagnosis, allergy work-up, management, treatment, and prevention of DRESS syndrome in light of currently available scientific evidence and the experience of experts from multiple disciplines., Methods: These guidelines were drafted by a panel of allergy specialists from the Drug Allergy Committee of the Spanish Society of Allergy and Clinical Immunology (SEAIC), together with other medical specialists involved in the management of DRESS syndrome and researchers from the PIELenRed consortium. A review was conducted of scientific papers on DRESS syndrome, and the expert panel evaluated the quality of the evidence of the literature and provided grades of recommendation. Whenever evidence was lacking, a consensus was reached among the experts., Results: The first Spanish guidelines on DRESS syndrome are now being published. Important aspects have been addressed, including practical recommendations about clinical diagnosis, identification of the culprit drug through the Spanish pharmacovigilance system algorithm, and the allergy work-up. Recommendations are provided on management, treatment, and prevention. Algorithms for the management of DRESS in the acute and recovery phases have been drawn up. Expert consensus-based stepwise guidelines for the management and treatment of DRESS syndrome are provided.

The levels of toxic metals (Al, Cd, Pb) and trace metals (B, Ba, Cu, Fe, Mn, Sr, Zn) were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) in the muscle (sirloin and chuck) and liver from a total of 180 samples of steers (less than 2 years old) (Bos taurus) of foreign and local origin slaughtered on the island of La Palma (Canary Islands, Spain). As regards toxic metals, Al was the metal with the highest contents in both tissues of the foreign steers (3.75 mg/kg in the muscle and 55.3 mg/kg in the liver) and the local steers (5.60 mg/kg in the muscle and 8.65 mg/kg in the liver). In conclusion, the present study confirmed that beef is a source of trace elements, mainly Fe and Zn. In addition, the consumption of this type of beef did not show significant intakes of toxic metals (Al, Cd, and Pb) and, therefore, the consumption of the steer muscle and liver does not pose a toxicological risk for Spanish consumers.

Marine pollution is one of today's most relevant problems. Public awareness has been raised about the harmful potential of heavy metals (HMs) accumulating in edible fish and possibly ending up in human diet through the food chain. This study aimed to characterize and evaluate As, Cd, Cr, Cu, Ni and Pb contents in four edible fish species from the western Mediterranean Sea. Liver and muscle toxic elements were determined by GF-AAS in Mullus surmuletus, Merluccius merluccius, Auxis rochei and Scomber japonicus from Almería Bay (Spain). Muscular composition, biometrics and trophic levels were also determined. The mean PTE concentration levels (mg kg -1 , DW) in fish muscle tissue were: As (2.90-53.74), Cd (0.01-0.18), Cr (0.53-2.01), Cu (0.78-6.93), Ni (0.06-0.24), Pb (0.0-0.32). These concentrations did not exceed the maximum limits set by European legislation (Commission Regulation (EC) No. 1881/2006) for the intake of these marine species. Accumulation of toxic elements tends to be seen in the liver (As (7.31-26.77), Cd (0.11-8.59), Cr (0.21-2.94), Cu (2.64-16.90), Ni (0.16-1.03), Pb (0.0-0.99)). As was the element at highest risk in this Mediterranean region, especially due to red mullet values in muscle. The high As contents with living habits as benthic species that feed near the coast. HMs, especially muscle Cd contents, were associated with higher contents of lipids and organic matter, and bigger specimen size (length and weight), while As was linked to higher fish protein content. However, these relationships between potentially toxic elements (PTE) and biometric indices and body composition parameters depend on species. Finally, the THQ indices indicated that eating fish from Almería Bay poses no human health risk despite pollution from the Almería coastline., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biochemical indicators and biomarkers were analyzed in the liver and gills of chub caught in three localities along the Sava River exposed to different environmental impacts. Sampling sites were: downstream from Zagreb (Zgd), downstream Sremska Mitrovica (SM) and downstream from Belgrade (Bgd). We observed that the relative amounts and levels of activity of Cu, Zn containing superoxide dismutase and glutathione in both the liver and gills, and the relative amounts of heat shock protein (HSP90) and metallothioneins in the gills were highest in the Zgd locality, suggesting a higher impact of metal pollution. The Zgd locality had higher concentrations of trace metals in the water, especially iron. In the SM and Bgd localities, higher relative levels of glutathione peroxidase and catalase were recorded (especially in SM) as compared to the Zgd locality, pointing to the presence of hydrogen peroxide and different classes of organic peroxides. Low water oxygen and high temperature levels in the Bgd locality suggesting different metabolic activity between examined locations. Our results suggest that different presence and concentrations of individual environmental factors (total environment) influence the way how fish establish homeostasis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Liver and gonad histopathology, biometric parameters and hepatic metal bioaccumulation were assessed monthly over a one-year period in common soles from the Basque continental shelf, in order to determine baseline levels and confounding factors within biomonitoring studies. Biometric parameters and hepatic metal bioaccumulation varied according to season and gender. Accordingly, hepatic histopathological traits presented seasonal variations related to the reproductive cycle. However, the hepatic histopathological index showed that seasonality and gender were not significant confounding factors. Conversely, the gonad histopathological index was modulated by season and gender. As for organ comparison, the liver endured more severe histopathological damage than the gonad. In brief, the sampling period and gender may not affect the estimation of hepatic histopathological indices for biomonitoring purposes. Nonetheless, due to different sensitivities to environmental 'noise' variables, the sampling period and gender differentiation should be thoroughly considered for the assessment of gonad histopathology, biometrics and metal bioaccumulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This work aims to develop and integrate new -omics tools that would be applicable to different ecosystem types for a technological updating of environmental evaluations. We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify/quantify proteins differentially expressed in the liver of free-living Mus spretus mice from Doñana National Park or its proximities. Mass spectrometry was performed in an LTQ Orbitrap system for iTRAQ reporter ion quantitation and protein identification using a Mus musculus database as reference. A prior IEF step improved the separation of the complex peptide mixture. Over 2000 identified proteins were altered, of which 118 changed by ≥2.5-fold in mice from at least two problem sites. Part of the results obtained with the iTRAQ analysis was confirmed by Western blot. Over 75% of the 118 proteins were upregulated in animals captured at polluted sites and only 16 proteins were downregulated. Upregulated proteins were involved in stress response; cell proliferation and apoptosis; signal transduction; metastasis or tumour suppression; xenobiotic export or vesicular trafficking; and metabolism. The downregulated proteins, all potentially harmful, were classified as oncoproteins and proteins favouring genome instability. The iTRAQ results presented here demonstrated that the survival of hepatic cells is compromised in animals living at polluted sites, which showed deep alterations in metabolism and the signalling pathways. The identified proteins may be useful as biomarkers of environmental pollution and provide insight about the metabolic pathways and/or physiological processes affected by pollutants in DNP and its surrounding areas., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aim: To explore the usefulness of magnetic resonance imaging (MRI) and spectroscopy (MRS) for assessment of non-alcoholic fat liver disease (NAFLD) as compared with liver histological and metabolomics findings., Methods: Patients undergoing bariatric surgery following procedures involved in laparoscopic sleeve gastrectomy were recruited as a model of obesity-induced NAFLD in an observational, prospective, single-site, cross-sectional study with a pre-set duration of 1 year. Relevant data were obtained prospectively and surrogates for inflammation, oxidative stress and lipid and glucose metabolism were obtained through standard laboratory measurements. To provide reliable data from MRI and MRS, novel procedures were designed to limit sampling variability and other sources of error using a 1.5T Signa HDx scanner and protocols acquired from the 3D or 2D Fat SAT FIESTA prescription manager. We used our previously described (1)H NMR-based metabolomics assays. Data were obtained immediately before surgery and after a 12-mo period including histology of the liver and measurement of metabolites. Values from (1)H NMR spectra obtained after surgery were omitted due to technical limitations., Results: MRI data showed excellent correlation with the concentration of liver triglycerides, other hepatic lipid components and the histological assessment, which excluded the presence of non-alcoholic steatohepatitis (NASH). MRI was sufficient to follow up NAFLD in obese patients undergoing bariatric surgery and data suggest usefulness in other clinical situations. The information provided by MRS replicated that obtained by MRI using the -CH3 peak (0.9 ppm), the -CH2- peak (1.3 ppm, mostly triglyceride) and the -CH=CH- peak (2.2 ppm). No patient depicted NASH. After surgery all patients significantly decreased their body weight and steatosis was virtually absent even in patients with previous severe disease. Improvement was also observed in the serum concentrations of selected variables. The most relevant findings using metabolomics indicate increased levels of triglyceride and monounsaturated fatty acids in severe steatosis but those results were accompanied by a significant depletion of diglycerides, polyunsaturated fatty acids, glucose-6-phosphate and the ATP/AMP ratio. Combined data indicated the coordinated action on mitochondrial fat oxidation and glucose transport activity and may support the consideration of NAFLD as a likely mitochondrial disease. This concept may help to explain the dissociation between excess lipid storage in adipose tissue and NAFLD and may direct the search for plasma biomarkers and novel therapeutic strategies. A limitation of our study is that data were obtained in a relatively low number of patients., Conclusion: MRI is sufficient to stage NAFLD in obese patients and to assess the improvement after bariatric surgery. Other data were superfluous for this purpose.

Objectives: The aim of the study was to investigate liver fibrosis outcome and the risk factors associated with liver fibrosis progression in hepatitis C virus (HCV)/HIV-coinfected patients., Methods: We prospectively obtained liver stiffness measurements by transient elastography in a cohort of 154 HCV/HIV-coinfected patients, mostly Caucasian men on suppressive antiretroviral treatment, with the aim of determining the risk for liver stiffness measurement (LSM) increase and to identify the predictive factors for liver fibrosis progression. To evaluate LSM trends over time, a linear mixed regression model with LSM level as the outcome and duration of follow-up in years as the main covariate was fitted., Results: After a median follow-up time of 40 months, the median increase in LSM was 1.05 kPa/year [95% confidence interval (CI) 0.72-1.38 kPa/year]. Fibrosis stage progression was seen in 47% of patients, and 17% progressed to cirrhosis. Aspartate aminotransferase (AST) levels and liver fibrosis stage at baseline were identified as independent predictors of LSM change. Patients with F3 (LSM 9.6-14.5 kPa) or AST levels ≥ 64 IU/L at baseline were at higher risk for accelerated LSM increase (ranging from 1.45 to 2.61 kPa/year), whereas LSM change was very slow among patients with both F0-F1 (LSM  ≤ 7.5 kPa) and AST levels ≤ 64 IU/L at baseline (0.34 to 0.58 kPa/year). An intermediate risk for LSM increase (from 0.78 to 1.03 kPa/year) was seen in patients with F2 (LSM 7.6-9.5 kPa) and AST baseline levels ≤  64 IU/L., Conclusions: AST levels and liver stiffness at baseline allow stratification of the risk for fibrosis progression and might be clinically useful to guide HCV treatment decisions in HIV-infected patients., (© 2014 British HIV Association.)

Objective: Fibroblast growth factor (FGF)-21, and possibly FGF19, protect against type 2 diabetes mellitus (T2DM) and obesity in rodents. We investigated the circulating levels of FGF21 and FGF19 in obese patients with varying degrees of abnormal glucose homeostasis, and we determined gene expression for FGF receptors (FGFR1-4) and the co-receptor β-Klotho, in liver and adipose tissues., Subjects and Methods: We analyzed 35 lean healthy (71% men) and 61 obese patients (49% men, median body mass index (BMI): 40.5 kg m(-2), interquartile range: 34.7-46.2). Among obese patients, 36 were normoglycemic, 15 showed impaired glucose tolerance and 10 had T2DM. Biopsies from liver and visceral and subcutaneous fat from a subset of obese patients and controls were analyzed. FGF19 and FGF21 levels were measured using enzyme-linked immunosorbent assay, and tissue mRNA and protein levels by reverse transcription-polymerase chain reaction and immunoblotting., Results: FGF21 serum levels were significantly increased in obese patients compared with controls (P<0.001), whereas FGF19 levels were decreased (P < 0.001). FGF21 levels were positively correlated with homeostasis model assessment of insulin resistance (P = 0.0002, r = 0.37) and insulin (P = 0.001, r = 0.32), whereas FGF19 levels were negatively correlated (P = 0.007, r = -0.27; P=0.003, r = -0.28; respectively). After adjusting for BMI, the correlations of FGF21 and FGF19 levels with indicators of abnormal glucose homeostasis were not significant. In obese patients, the hepatic expression of FGF21 was increased. (P = 0.04). β-Klotho transcript levels in visceral fat (P = 0.002) and β-Klotho protein levels in subcutaneous (P = 0.03) and visceral fat (P = 0.04) were significantly reduced in obese patients, whereas hepatic levels for β-Klotho (P = 0.03), FGFR1 (P = 0.04) and FGFR3 (P = 0.001) transcripts were significantly increased., Conclusions: Obesity is characterized by reciprocal alterations in FGF19 (decrease) and FGF21 (increase) levels. Although worsened in diabetic obese patients, obesity itself appears as the predominant determinant of the abnormalities in FGF21 and FGF19 levels. Opposite changes in β-Klotho expression in fat and liver indicate potential tissue-specific alterations in the responsiveness to endocrine FGFs in obesity.

Objectives: The objective of our follow-up study is to evaluate liver function tests (LFTs) and lipid profiles in patients with Gilbert's syndrome treated with isotretinoin because of severe acne., Setting: Dermatology outpatient clinics of three regional hospitals of Jaén (Spain)., Participants: Over 4 years, we included all patients diagnosed with severe acne. Only 37 patients were identified, of which 11 had Gilbert's syndrome., Interventions: All patients were treated with isotretinoin and followed-up in our outpatient clinics after 10 and 20 weeks. Patients were subjected to an interview questionnaire which included data on age, gender, complete blood count, coagulation profile, fasting blood glucose, LFTs and lipid profiles. Data and results of patients with severe acne and Gilbert's syndrome were compared with those of 26 patients with only severe acne (control group)., Primary Outcome: Blood analyses were repeated in the follow-up visits., Results: In patients with Gilbert's syndrome, bilirubin levels showed substantial decrease over the 20-week follow-up, with more decrease after 10 weeks. None of the control group patients had significant increase in total bilirubin levels after 10 and 20 weeks of follow-up. Liver enzymes were maintained within normal levels in both groups. Both study groups did not show significant pathological increase in lipid profile levels. LDL levels were increased in the two study groups, but this increase was less substantial in patients with Gilbert's syndrome., Conclusions: Our preliminary results suggest that oral isotretinoin could be an effective, safe treatment for patients with Gilbert's syndrome, and may lower bilirubin levels in the first 10 weeks of treatment. Limitations of the study include the small numbers of participants and the fact that it is restricted to one region of Spain.

Seven deep-sea fish species were sampled in the Blanes Canyon area (NW Mediterranean) at a depth of 1200 m during winter. The concentrations of nine metals were determined in the liver of these species by ICP-MS. Furthermore, the metal detoxification potential was determined for each species by analysing the hepatic metallothionein (MT) content, relations between metals and the molar ratio between MT and/or selected metals. The potential effect of metal content on their physiology was assessed using general stress markers such as the enzyme activities of acetylcholinesterase (AChE) and lactate dehydrogenase (LDH) in muscle. Levels of metals in the seven Mediterranean deep-sea fish species studied were intermediate to equivalent species of fish either from Atlantic waters or hydrothermal vents. The metal detoxification potential varied among species depending on MT, selenium (Se) or zinc (Zn) as reliable mechanisms to handle potential metal toxicity. The role of Se was especially relevant when the liver content of mercury (Hg) was higher. AChE and LDH activities did seem to be affected by metal loads and thus the activities reported would correspond to baseline activities of the selected species., (© 2013.)

Metabolic activation of pesticides in the liver may result in highly reactive intermediates capable of impairing various cellular functions. Nevertheless, the knowledge about the effect of pesticide exposure on liver function is still limited. This study assessed whether exposure to pesticides elicits early biochemical changes in biomarkers of liver function and looked for potential gene-environmental interactions between pesticide exposure and polymorphisms of pesticide-metabolizing genes. A longitudinal study was conducted in farm-workers from Andalusia (South Spain), during two periods of the same crop season with different degree of pesticide exposure. Blood samples were taken for the measurement of serum and erythrocyte cholinesterase activities as well as for determining clinical chemistry parameters as biomarkers of liver function. Serum lipid levels were also measured as they may help to monitor the progress of toxic liver damage. A reduction in serum cholinesterase was associated with decreased levels of all clinical chemistry parameters studied except HDL-cholesterol. Conversely, a decreased erythrocyte cholinesterase (indicating long-term pesticide exposure) was associated with increased levels of aspartate aminotransferase and alkaline phosphatase and increased levels of triglycerides, total cholesterol and LDL-cholesterol, but reduced levels of HDL-cholesterol. Changes in liver biomarkers were particularly associated with the PON155M/192R haplotype. The obtained results therefore support the hypothesis that pesticide exposure results in subtle biochemical liver toxicity and highlight the role of genetic polymorphisms in pesticide-metabolizing enzymes as biomarkers of susceptibility for developing adverse health effects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Recent studies linked circulating pigment epithelium-derived factor (PEDF) to obesity-associated insulin resistance, but the main source of circulating PEDF is unknown. We aimed to investigate liver and adipose tissue PEDF gene expression in association with obesity and insulin resistance., Design, Subjects and Methods: Three (two cross-sectional and one longitudinal) independent cohorts have been studied, for adipose tissue (n=80 and n=30) and liver gene expression (n=32 and n=14). Effects of high glucose and cytokines on HepG2 cell line were also investigated. PEDF gene expression and circulating PEDF were analyzed using real-time PCR and ELISA, respectively., Results: In a first cohort of subjects, PEDF relative gene expression was higher in subcutaneous (SC) than in omental (OM) adipose tissue (P<0.0001) being also higher in mature adipocytes compared with stromo-vascular cells (P<0.0001). However, OM PEDF relative gene expression was decreased in morbidly obese subjects (P=0.01). Both OM PEDF and OM PEDF receptor (PEDFR) correlated positively with lipogenic and lipolytic genes, and with genes implicated in the lipid vacuole formation. Circulating PEDF levels were not associated with fat PEDF gene expression. In the second cohort, SC PEDF was decreased in subjects with type 2 diabetes and did not change significantly after weight loss. We next explored circulating PEDF in association with markers of liver-related insulin resistance injury (alanine aminotransferase, r=0.59, P=0.001). Interestingly, liver PEDF gene expression increased with obesity and insulin resistance in men, being significantly associated with fasting glucose and glycated hemoglobin in two independent cohorts. In fact, high glucose led to increased PEDF in HepG2 cells, while inflammatory stimuli present in the adipose tissue environment downregulated PEDF., Conclusion: Liver, but not adipose tissue, might be the source of increased circulating PEDF linked to insulin resistance.

Background: Procyanidins are extensively metabolized via phase-II and microbial enzymes. However, their distribution in the body is not well characterized., Aim: This study investigates the distribution of procyanidins (monomers and dimers) and their phase-II metabolites in plasma and tissues (thymus, heart, liver, testicle, lung, kidney, spleen and brain)., Methods: Wistar rats were fed with 1 g of cocoa cream (CC), 50 mg of procyanidin hazelnut skin extract (PE) and 50 mg PE in 1 g CC (PECC). The rats were killed at 0, 1, 1.5, 2, 3, 4 and 18 h after gavage, and the plasma and tissues were analyzed by UPLC-MS/MS., Results: Epicatechin-glucuronide was the main metabolite in the plasma after the CC intake, with C(max) at 423 nM and t(max) at 2 h, and methyl catechin-glucuronide (301 nM, 2 h) was the main metabolite in the plasma after the PE intake. As a result of the PECC enrichment, epicatechin-glucuronide (452 nM, 1.5 h) and catechin-glucuronide (297 nM, 2 h) were the main metabolites in the plasma. Methyl catechin-glucuronide was found in the liver after PE (8 nmol/g tissue, 4 h) and PECC (8 nmol/g, 1.5 h). The kidney was found to contain a high concentration of phase-II metabolites of procyanidins and is therefore thought to be the main site of metabolism of the compounds. Methyl catechin-sulfate (6.4 nmol/g, 4 h) was only quantified in the brain and after PE intake. Catechin metabolites were not found in the spleen or heart. Phenolic acids were detected in all tissues., Conclusions: The formulation of a product enriched or fortified with procyanidins is a way to increase their bioavailability, with clear effects on the plasmatic pharmacokinetics, and a greater accumulation of phenolic metabolites in such tissues as the liver, kidney, lung and brain.

Low-fat pork liver pâtés enriched with n-3 PUFA/konjac gel were formulated by replacing (totally or partially) pork backfat by a combination of healthier oils (olive, linseed and fish oils) and konjac gel. Lipid oxidation, microbiological changes and biogenic amine (BA) formation were studied in healthier-lipid pâtés during chill storage (85 days, 2 °C). Increasing unsaturated fatty acid levels favoured lipid oxidation, although the levels reached were low throughout the storage period, ranging from 0.113 to 0.343 mg malonaldehyde/kg sample. Neither the formulation nor the time in storage affected the microbial load. Biogenic amine contents of products (the sum of initial concentrations and amines formed during storage) varied according to the type of BA but were far below levels that could constitute a consumer health hazard., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme catalyses the regeneration of active cortisol from inert cortisone and plays a critical role in tissue-specific corticosteroid reactions; therefore, 11β-HSD1 is a key molecule associated with the development of obesity. Despite evidence for its role in obesity, no genetic polymorphisms have been significantly associated with the disease per se., Objective: The aim of this study was to evaluate whether HSD11B1 gene variants, which have never been studied before, are associated with obesity and its related traits, as well as its relation to biomarkers of inflammation, liver damage and cardiovascular disease in a cohort of Spanish children., Design: We performed a prospective case-control study., Subjects: A total of 534 children were examined and classified as being obese (n=292) or normal weight (n=242). Anthropometric and biochemical measurements related to obesity, including inflammation, liver damage and cardiovascular disease, were determined. Genomic DNA was extracted and 10 HSD11B1 gene single-nucleotide polymorphisms (SNPs) were genotyped., Results: A novel SNP, rs3753519, was strongly associated with obesity and this SNP was the only statistically significant HSD11B1 gene SNP remaining after a Bonferroni correction (odds ratio=1.97 for allelic effect, 95% confidence interval 1.23-3.16; P=0.004 and Bonferroni corrected P=0.046). In addition, this SNP was significantly and positively associated with increased body mass index (BMI), BMI z-score, weight, waist circumference, plasma γ-glutamyl transpeptidase and plasma active plasminogen activator inhibitor 1. The SNP was negatively associated with plasma adiponectin and cortisol after adjusting for sex and age. None of the inflammation biomarkers tested were associated with the risk allele., Conclusion: These data, which link an HSD11B1 genotype with both disease prevalence and its related phenotypes, strongly support a role for the rs3753519 polymorphism in the pathogenesis of pediatric-onset obesity.

A metallomic approach based on the use of size-exclusion chromatography (Superdex-75) with inductively coupled plasma mass spectrometry (ICP-MS) detection is combined with anion or cation exchange chromatography to characterize the biological response of the free-living mouse Mus spretus. The approach has been applied to contaminated and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas affected by differential contamination from mining, industrial, and agricultural activities have been considered. The high presence of Mn, Cu, and Zn in liver and As and Cd in kidney is remarkable, especially in contaminated areas. The size exclusion chromatograms traced by Mn in liver cytosolic extracts are more intense than in kidney; a Mn-peak matching with the standard of 32 kDa (superoxide dismutase) is present in these organs, and its intensity is correlated with the concentration of Mn in the extracts. High-intensity peaks traced by Cu, Zn, and Cd at 7 kDa (matching with metallothionein I standard) in liver extract are triggered by the presence of contaminants. Other peaks related with molecules of 32 and 67 kDa traced by Cu and Zn can also be observed, although their intensity is higher in sites with low contamination. In kidney extracts, the presence of a Cd-peak with Mr of 7 kDa (tentatively Cd-metallothionein) with high intensity under the action of contaminants was observed, but high biological responses are also proven in the protected area of the Park, which denotes a progressive increase of diffuse contamination.

The aim of this paper is to evaluate at a histopathological level the effect of the most commonly used copper (Cu) supplementation (15 mg/kg dry matter (DM)) in the liver of intensively reared beef cattle. This was done by a histochemistry evaluation of (i) the antioxidant capacity in the liver - by the determination of metallothioneins (MT) and superoxide dismutase (SOD) expression - as well as (ii) the possible induction of oxidative damage - by the determination of inducible nitric oxide synthase (iNOS), nitrotyrosine (NITT), malondialdehyde (MDA) and 8-oxoguanine (8-oxo) - that (iii) could increase apoptotic cell death - determined by cytochrome-c (cyto-c), caspase 1 (casp1) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Liver samples from Cu-supplemented (15 mg Cu sulphate/kg DM, n = 5) and non-supplemented calves (n = 5) that form part of other experiments to evaluate Cu status were collected at slaughter and processed for immunohistochemistry and TUNEL. MT expression was diffuse and SOD showed slight changes although without statistical significance. iNOS and NITT positive (+) cells significantly increased, mainly around the central veins in the animals from the Cu-supplemented group, whereas no differences were appreciated for the rest of the oxidative stress and apoptosis markers. Under the conditions of this study, which are the conditions of the cattle raised in intensive systems in NW Spain and also many European countries, routinely Cu supplementation increased the risk of the animals to undergo subclinical Cu toxicity, with no significant changes in the Cu storage capacity and the antioxidant defensive system evaluated by MT and SOD expression, but with a significant and important increase of oxidative damage measured by iNOS and NITT. The results of this study indicated that iNOS and NITT could be used as early markers of initial pathological changes in the liver caused by Cu supplementation in cattle, although more studies in cattle under different levels of Cu supplementation are needed.

To determine possible mechanisms of action that might explain the nutrient partitioning effect of betaine and conjugated linoleic acid (CLA) in Iberian pigs and to address potential adverse effects, twenty gilts were restrictively fed from 20 to 50 kg BW Control, 0.5% betaine, 1% CLA or 0.5% betaine + 1% CLA diets. Serum hormones and metabolites profile were determined at 30 kg BW and an oral glucose test was performed before slaughter. Pigs were slaughtered at 50 kg BW and livers were obtained for chemical and histological analysis. Decreased serum urea in pigs fed betaine and betaine + CLA diets (11%; P = 0.0001) indicated a more efficient N utilization. The increase in serum triacylglycerol (58% and 28%, respectively; P = 0.0098) indicated that CLA and betaine + CLA could have reduced adipose tissue triacylglycerol synthesis from preformed fatty acids. Serum glucose, low-density lipoprotein (LDL) cholesterol and non-esterified fatty acids were unaffected. CLA and betaine + CLA altered serum lipids profile, although liver of pigs fed CLA diet presented no histopathological changes and triglyceride content was not different from Control pigs. Compared with controls, serum growth hormone decreased (20% to 23%; P = 0.0209) for all treatments. Although serum insulin increased in CLA, and especially in betaine + CLA pigs (28% and 83%; P = 0.0001), indices of insulin resistance were unaffected. In conclusion, CLA, and especially betaine + CLA, induced changes in biochemical parameters and hormones that may partially explain a nutrient partitioning effect in young pigs. Nevertheless, they exhibited weak, although detrimental, effects on blood lipids. Moreover, although livers were chemically and histologically normal, pigs fed CLA diet challenged with a glucose load had higher serum glucose than controls.

The Spanish imperial eagle (Aquila adalberti) is found only in the Iberian Peninsula and is considered one of the most threatened birds of prey in Europe. Here we analyze lead concentrations in bones (n = 84), livers (n = 15), primary feathers (n = 69), secondary feathers (n = 71) and blood feathers (n = 14) of 85 individuals collected between 1997 and 2008 in central Spain. Three birds (3.6%) had bone lead concentration > 20 μg/g and all livers were within background lead concentration. Bone lead concentrations increased with the age of the birds and were correlated with lead concentration in rachis of secondary feathers. Spatial aggregation of elevated bone lead concentration was found in some areas of Montes de Toledo. Lead concentrations in feathers were positively associated with the density of large game animals in the area where birds were found dead or injured. Discontinuous lead exposure in eagles was evidenced by differences in lead concentration in longitudinal portions of the rachis of feathers.

The concentrations of cadmium, mercury and lead were determined in liver, kidney and feathers of Accipiter gentilis, Buteo buteo and Strix aluco. No significant differences in the concentrations of metals were observed in relation to either age or gender. Cadmium levels in feathers and in kidney were significantly correlated (p < 0.01 or p < 0.05) in all species. In B. buteo, cadmium concentrations in feathers and in liver were also correlated (p < 0.01), as were the concentrations of lead in feathers and in liver (p < 0.05). The low correlation coefficients let to reject the possibility of predicting metals concentrations in viscera from the concentrations in feathers.

In animals, exposure to metal pollution can induce oxidative stress via several mechanisms. This stress might then cause adverse effects on functions such as male reproductive capacity. Antioxidant vitamins A and E play an important role in maintaining organism functions under stressed conditions. This study assessed the effect of different metals and metalloids on levels of vitamins A and E in livers and testis (n = 67 and 36) of red deer and in livers (n = 22) of wild boar. The study compared animals residing within and outside a polluted mining area. Red deer from mined areas showed significant reductions in liver retinyl docosahexaenoate and retinyl docosapentaenoate. Free retinol, α-tocopherol, and retinyl palmitate in the testis were also lower. This might indicate that increased internal usage of these antioxidants is occurring as deer try to maintain the integrity and function of reproductive tissue. Wild boar from mined areas also showed significant reductions in liver retinyl stearate but increased free retinol levels. This might suggest that vitamin A is being mobilized to a greater degree to cope with the induced oxidative stress caused by exposure to metal pollution. Additionally, a significant negative relationship between liver α-tocopherol and bone lead (Pb) in boar might indicate some long-term effects of Pb on antioxidant levels. Results suggest that vitamin A and E status can be altered as a consequence of exposure to Pb pollution and that complex differences in this response probably exist between species.

In the present study, bioaccumulation of lead, cadmium and zinc in liver, kidney and muscle of roe deer (Capreolus capreolus) from Galicia (NW Spain) was quantified. Metal analysis was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). In general the quantified concentrations were associated with background environmental levels, except for Pb, where some markedly elevated concentrations were obtained, but with no toxicological relevance. The effect of sex on Cd, Pb and Zn levels was considered, females showing a general trend to accumulate higher concentrations of these metals, with the exception of the hepatic content of Cd (which was significantly higher in males, P < 0.05) and Pb levels, which did not differ according to sex. The effect of age was analysed only in male animals and was clearly observed for Pb levels, with concentrations significantly higher in adult animals (liver: P < 0.01, kidney: P < 0.05 and muscle: P < 0.001) and for Cd levels in kidney samples (P < 0.001). This study provides the first data on heavy metal content in roe deer from the area of study and suggests that this species could be a good bioindicator of metal pollution, even though both factors, sex and age, could represent an important source of variation in the bioaccumulation of these metals in wild populations.

The effluents from sewage treatment plants strongly influence the water quality and flow of Mediterranean streams. These effluents play a crucial role in maintaining the aquatic communities of these ecosystems, particularly in the absence of natural flow resulting from climate constraints or intensive water use. To detect the ecological effects of these effluents, we used non-lethal biomarkers in Barbus meridionalis and traditional assessment protocols in the Ripoll River (NE of Spain). Our results demonstrate the utility of haematological parameters. In comparison with fish at reference sites, we detected a decrease in haematocrit and haemoglobin, neutrophilia, lymphopenia, monocytosis, a rise in the nucleo-cytoplasmatic ratio of erythrocytes and an increase in the frequency of abnormal, immature and senescent erythrocytes. Many haematological parameters correlated significantly with the environmental parameters measured. In addition to these haematological alterations, histopathological examination also revealed damage in fish livers but no impact was detected by the regional index of biotic integrity using fish as bioindicators. To the best of our knowledge, this study is the first to report this battery of haematological parameters as biomarkers on a freshwater fish in a Mediterranean stream. In a region in which more than 50% of native fish species are classified as endangered or vulnerable, there is an urgent need to find biomarkers that do not harm the animals., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Pollution in riverine systems, along with its biological effects, may propagate downstream even at considerable distances. We analyzed the organochlorine compound (OC) pollution in a section of the low Ebro River (Northeast Spain) downstream a long-operating chlor-alkali plant. Maximal levels of OCs and of their associated dioxin-like biological activity occurred in residue samples from the plant, and persisted in river sediments some 40km downstream (Xerta site). Biological analysis at multiple organization levels in local carp (Cyprinus carpio, EROD, Cyp1A mRNA expression in the liver, hepatosomatic index, condition factor, and micronuclei index in peripheral blood) showed a similar pattern, with a maximal impact in Ascó, few kilometers downstream the plant, and a clear reduction at Xerta. This combination of chemical, molecular, cellular and physiological data allowed the precise assessment of the negative impact of the chlor-alkali plant on the quality of river sediments and on fish, and suggests that sediments may be a reservoir for toxic substances even in dynamic environments like rivers., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: Genetic and dietary hyperhomocysteinemia has been found to decrease high density lipoproteins (HDL) and their apolipoprotein A1 (APOA1). To test the hypothesis that the presence of cysteine could normalize HDL levels in hyperhomocysteinemic cystathionine beta-synthase (Cbs)-deficient mice and that the inclusion of glycine would block this effect., Methods: Lipids and HDL cholesterol were studied in Cbs-deficient mice and wild-type animals fed a low-methionine diet supplemented with cysteine and glycine and in Cbs-deficient mice on the same diet supplemented only with cysteine., Results: Triglyceride and homocysteine levels were significantly decreased and increased, respectively in Cbs-deficient mice irrespective of treatment. However, plasma cholesterol, glucose and APOA1 were significantly decreased in homozygous Cbs-deficient mice when they received the cysteine and glycine-enriched beverage. This group of mice also showed decreased mRNA levels and increased hepatic content of APOA1 protein, the latter increase was observed in endothelial cells. A significant, inverse relationship was observed between plasma and hepatic APOA1 concentrations while a positive one was found between plasma levels of cysteine and APOA1., Conclusion: These data suggest an altered hepatic management of APOA1 and that cysteine may be involved in the control of this apolipoprotein at this level. Overall these findings represent a new aspect of dietary regulation of HDL at the hepatic transendothelial transport., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor alpha (pparalpha) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARalpha mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor alpha was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: The significance of H63D homozygosity remains uncertain, although it is associated with a tendency for patients to develop iron overload., Aims: To study the prevalence of homozygotic H63D mutation in patients with phenotypic hemochromatosis (PH) and to compare the results with those of the general population and with patients with porphyria cutanea tarda (PCT) in the Basque Country, Spain. A secondary aim was to evaluate the differences in phenotypic expression and liver injury according to different genotypes in the PH cohort., Methods: Mutations of the HFE gene were obtained by polymerase chain reaction (PCR). Forty consecutive patients diagnosed with PH, 116 controls and 54 patients with PCT were included in the study. We performed liver biopsies, measured liver iron concentration (LIC), by atomic spectrophotometry, serum ferritin and transferrin saturation, and compared the histology according to the genotype., Results: The H63D homozygote mutation was identified in 7.76% of the control group, in 7.50% of the PH group, and in 11.11% of patients with PCT (P > 0.05). The C282Y/C282Y mutation was present in 50% of patients with PH, and LIC was identified in 15/20. The LIC in C282Y/C282Y patients was higher than in H63D/H63D patients (P = 0.26), while H63D homozygosis caused greater iron overload in PH patients than other genotypes. All the C282Y/C282Y genotype patients had elevated serum ferritin and transferrin saturation. The H63D homozygotes had high ferritin, but two out of three had normal transferrin saturation. Six of the eight patients with high-grade fibrosis and genetic study results were found to be C282Y/C282Y., Conclusions: The prevalence of H63D mutation in patients with PH in our region does not differ from that of the general Basque population.

Sea turtles are of increasing interest as potential bioindicators of the heavy metal pollution in marine ecosystems. In the present work, concentrations of heavy metals and essential elements (As, Cd, Hg, Pb, Se, Zn) in different organs and tissues (liver, kidney, muscle, bone, blood, central nervous system and skin) of loggerhead sea turtles (Caretta caretta) were determined from stranded animals found along the Spanish Mediterranean coastlines of Murcia. Relatively high average levels of As (skin: 52.13 microg g(-1) dry weight; muscle: 40.95 microg g(-1) dry weight), and especially high individual levels of Zn in muscle tissue (1002.4 microg g(-1) dry weight) were detected. Furthermore, a significant degree of organotrophism of Cd was observed in kidney tissue. The concentrations detected, the distribution among the tissues and the differences observed between juvenile and adult specimens are generally compatible with chronic exposure to the elements studied, whilst levels produced by acute exposure were ruled out., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The Pb mining area of the valley of Alcudia and the Sierra Madrona mountains (S. Spain) has been exploited intermittently for over 2100 years, since Roman occupation and up until the late 1900s. Red deer (n=168) and wild boar (n=58) liver and bone (metacarpus) were analyzed for Pb, Zn, Cu, Cd, As and Se. Lipid peroxidation, total and oxidized glutathione (GSH), superoxide dismutase (SOD), GSH peroxidase (GPX) and fatty acid composition were studied in liver of red deer. The concentrations of Pb in liver and bone of red deer and wild boar were higher in the mining area than in the control area, and higher in the wild boar than red deer, but well below the level associated with clinical signs of Pb poisoning. Liver levels of Cu, Cd and Se were also higher in red deer from the mining area. Red deer from the mining area had 39% less total GSH than in the control area. The percentage of docosahexaenoic acid in liver of red deer from the mining area was 16% lower than in the control area.

Nine fish species of commercial interest from six teleost families and two species of elasmobranchs were selected for characterisation of hepatic biomarkers used in early-warning assessment of pollutant exposure. The sampling was carried out in front of the Barcelona coast (NW Mediterranean) during December 2006 at shelf (53 m) and slope (660 m) depths. The enzymes considered included the antioxidant defence catalase (CAT) and glutathione reductase (GR), phase I ethoxyresorufin O-deethylase (EROD) and phase II glutathione S-transferase (GST). Protein yield (PY) was used as a general marker of hepatic protein synthesis. Significant interspecies differences were evidenced, although each marker varied independently. Enzymatic activities in teleosts were higher than in elasmobranchs; they were very low in Scyliorhinus canicula (mainly a benthic feeder), but not so low in Galeus melastomus (pelagic feeder). In relation to depth, shallow water, shelf-living species had higher metabolic activities. Trophic variables were significantly related to PY and EROD activity, and were especially high in benthic/suprabenthic feeders. Trophic level (deduced from stable isotopy) and stomach fullness were associated with all hepatic markers, except GR. Swimming capacity was related to all hepatic enzymes. Our findings can be applied, not only from the perspective of conservation ecology regarding pollution, but also in fisheries, due to the economic interest of the species involved.

Polychlorinated biphenyls (PCBs 28, 31, 52, 101, 138, 153, 180, and 209) were measured in tissue samples (liver and fat) from 30 loggerhead turtles Caretta caretta, 1 green turtle Chelonia mydas, and 1 leatherback Dermochelys coriacea stranded on the coasts of the Canary Islands, trying to establish a possible relation between PCB concentrations and the lesions and causes of death. Tissues from these turtles contained higher levels of PCBs than those reported in turtles from other geographical regions. Sigma PCB concentrations (1980+/-5320 ng g(-1)wet wt.) in the liver of loggerheads were higher than in the adipose tissue (450+/-1700 ng g(-1)wet wt.). Concentrations of PCB 209 in the liver (1200+/-3120 ng g(-1)wet wt.) of loggerheads and in the liver (530 ng g(-1)wet wt.) and adipose tissue (500 ng g(-1)wet wt.) of the leatherback were remarkable. Frequencies of detection of PCB 209 in the liver (15.5%) and adipose tissue (31%) were also remarkable. Cachexia was detected in 7 turtles (22%) and septicemia was diagnosed in 10 turtles (31%). Statistically, a positive correlation was detected between Sigma PCBs concentration and cachexia. Poor physical condition, cachexia and/or septicaemia could explain the high levels of PCBs and tissue distribution. However, no histological lesions exclusively attributed to the acute effects of PCBs were described. The most prevalent histological lesions were ulcerative and purulent oesophagitis, purulent dermatitis, necrotizing enteritis, and granulomatous pneumonia. The bacteria most frequently isolated were Escherichia coli, Staphylococcus sp., and Aeromonas sp. Although immunosupression as a result of PCBs pollution has been described previously, other factors in this study, such as incidental fishing, nutritional status, and exposition to different micro-organisms, make it difficult to establish a clear association between PCB concentrations and causes of death.

The Prestige oil spill resulted in the mortality of several seabird species on the Atlantic NW coast of Spain. Shag casualties were particularly relevant, since populations are resident in the area the whole year round and because of several features which make them highly vulnerable to environmental hazards. Ecological catastrophes give us the opportunity of collecting samples which, otherwise, would be difficult to obtain. We examine the potential of shag corpses as bioindicators of inorganic pollution and the possible factors of variability, such as biological traits (sex, age) or nutrition status. We determined trace elements (Hg, Se, Cr, Pb, Zn and Cu) and isotopic signatures (15N, 13C) in soft tissues (muscle, liver) and in primary feathers formed at different times (before and after the Prestige) in individuals of known sex and age, collected at the time of the Prestige disaster. These were compared with data from another group of shags trapped accidentally in fishing gear in 2005. Our results did not seem to be affected by sex or age on any of the analysed variables. The higher nitrogen isotopic signatures in the soft tissues of the Prestige shags may be related to the nutrition stress caused by a poorer body condition, which is also reflected in increasing levels of some metals in the liver. This isotopic enrichment was also observed in newly forming feathers when compared to the old ones. On the other hand, the lower delta15N and Hg values in shag feathers from 2005 point to a shift in feeding resources to prey of lower trophic levels. We found that feather features (being an inert tissue and having a conservative composition), if combined with careful dating and chemical analysis, offer a very useful tool to evaluate temporal and spatial changes in seabird ecology in relation to pollution events.

Polybrominated diphenyl ethers (PBDEs) are chemicals of environmental concern due to their lipophilic, persistent and bioaccumulable characteristics as well as for their potential endocrine disrupting role. Former studies carried out in a tributary of the Cinca river (Ebro basin, NE Spain) revealed high levels of PBDEs in fish due to the discharges of effluents rich in PBDEs coming from a nearby industrial park in Barbastro. In this study, several biomarkers of pollutants exposure were measured in barbel, Barbus graellsii, before (upstream) and after (downstream) the main industrial site (Barbastro) in the Vero river. The results evidenced an enhanced hepatic phase I and II metabolism (measured as reductases, glutathione S transferase and uridinediphospho-glucuronosyltransferase), and of the antioxidant enzyme glutathione peroxidase. Conversely, fishes collected from downstream reaches had their phase I CYP1A dependent ethoxyresorufin O-deethylase, antioxidant diaphorase and brain cholinesterase activities depleted. In addition, the histological study of the liver and kidney of these fish evidenced an increase of the number and size of macrophage aggregates in most individuals collected downstream. Bivariate correlated analyses showed that the above mentioned biomarkers were correlated to measured PBDE congeners, thus indicating that the observed biological effects were unlikely to be related to other environmental factors than PBDEs. Overall, the measured biochemical and histological markers provide new evidence that in field exposed fish, PBDEs levels were associated with high activities of phase I and II metabolic enzymes, oxidative stress in liver, neurotoxicity in brain and histopathological effects in both liver and kidney.

The Iberian lynx (Lynx pardinus) is the most endangered felid in the world with a wild population which probably stands at less than 200 individuals inhabiting two areas in Southern Spain (Doñana and Sierra Morena) that are known to have been contaminated by heavy metals and metalloids due to a long history of mining activities. This contamination may pose a threat to long term conservation efforts and hence, the concentrations of seven elements (As, Se, Cd, Zn, Cu, Pb, Hg) were determined in the liver, muscle and bone of 9 lynx, as well as 17 red foxes (Vulpes vulpes), 11 Egyptian mongooses (Herpestes ichneumon), 4 common genets (Genetta genetta) and 1 Eurasian badger (Meles meles). The mean concentrations found were below the threshold levels indicative of chronic intoxication in all the species studied. In general, genet and red fox were species with the highest concentrations of several elements in Doñana, whilst Iberian lynx had the lowest levels of most of them. Lynx from Sierra Morena had significantly higher concentrations of bone Pb (2.05 microg/g d.w.) than those from Doñana (0.13 microg/g d.w.), probably due to the mineralised underlying geology and/or the abandoned mine workings in Sierra Morena. Egyptian mongoose presented liver concentrations of Hg up to 9.7 microg/g d.w. A strong relationship between Hg and Se levels was found in liver and muscle samples of all the studied species, especially in mongoose. In conclusion, levels of the studied elements do not appear to represent a significant threat for the lynx or for the other carnivores studied. However, given the critical status of the Iberian lynx, a continuous monitoring scheme remains necessary.

As top consumers in food chains, birds of prey forage over large geographical areas and so might be expected to accumulate environmental contaminants which are distributed in the environment. These wild animals can offer opportunities to detect and assess the toxicological effects of different inorganic elements on terrestrial ecosystems. In this study, different raptor species, both diurnal and nocturnal, were investigated for heavy metal (Pb, Cd and Zn) and As concentrations in liver samples, with the aim of furnishing indirect information concerning contamination of their habitats. Dead animals were obtained with the special collaboration of the Wildlife Rehabilitation Centres from Galicia (NW Spain). After sample wet digestion, metal analysis was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Hepatic concentrations of Zn and As, respectively, situated on the interval 147-298 and 1.21-6.88 ppm (dry weight, dw), could be considered as indicative of low and background amounts of both elements, with no ecotoxicological concern. Nevertheless, with respect to Pb, some diurnal raptors showed hepatic concentrations above the considered threshold value (6 ppm dw) for sublethal or lethal toxicity, the species with the highest hepatic level corresponding to a common buzzard (>18 ppm, dw). Similarly, nocturnal raptors exceeded the threshold value for Cd (3 ppm dw), with a maximum corresponding to an individual barn owl (39 ppm, dw). In both cases, although concentrations could not be directly related to lethal effect, they might constitute a serious environmental factor affecting the survival of the considered populations.

Polychlorinated biphenyls (PCBs) and chlorinated pesticides: dichlorodiphenyltrichloroethane and its metabolites (DDTs), chlordanes (CHLs), dieldrin, and hexaclorobenzene (HCB) were detected in the blubber and liver of 11 bottlenose dolphins (Tursiops truncatus) from the Canary Islands (North Atlantic Ocean). Samples were obtained from stranded dolphins over the period 1997-2005. Among the organochlorines analyzed, PCBs and DDTs were predominant in the two tissues, followed in decreasing order by chlordane, trans-nonachlor > cis-nonachlor > dieldrin and HCB. The sum 11 PCBs in the blubber ranged between 301 and 33,212 ng g(-1) ww (990 and 136,679 ng g(-1) lw). Highly chlorinated PCBs such us CB153, CB180, and CB138 were the prominent congeners, accounting for 51% of the total PCBs. The sum DDT concentration in the blubber ranged between 147 and 21,050 ng g(-1) ww. (490-105,250 ng g(-1) lw) The main DDT metabolite was p,p'-DDE, representing 83% of DDTs in the blubber. In general, the levels of PCBs and DDTs detected were similar to those found in bottlenose dolphins in the North of Europe. The 2,3,7,8-TCDD toxic equivalent (TEQ) in blubber and liver was calculated for the toxicity assessment of mono-ortho substituted PCBs congeners (CB105, CB118, CB156). It is important to mention that TEQ values and p,p'-DDE concentration in adult male specimens are approaching the levels associated with adverse effects found in marine mammals. The information provided represents the first tissue loads of organochlorine compounds in small cetaceans from this area., ((Copyright) 2008 Wiley Periodicals, Inc.)

Metal levels (Cd, Pb, Hg, Cr, Fe, Cu, Zn) together with metallothioneins (MTs) were determined in the liver of two commercial fish species collected along the Northern Iberian coast. The four-spotted megrim (Lepidorhumbos boscii) and the pouting (Trisopterus luscus) were selected as representative species of the middle/outer (200-500 m) and inner shelf (70-120 m), respectively. Metal pollution in the middle/outer shelf was mostly detected in the Asturias and Basque Country areas, whereas in the inner shelf, pollution was widely spread along the coast. Significantly high levels of Pb, Cd, Cr and Hg were detected in T. luscus from different sampling sites and associated to anthropogenic pressure. MTs were induced in fish that had higher amounts of Zn, Cd, Cr and Hg in their livers, supporting the use of MT as a biomarker of metal exposure. The study supports the usefulness of the selected fish species as sentinel organisms for future biomonitoring studies.

The study of the dynamics of phytobenthic and phytoplankton communities was undertaken, during a year, in the regulation reservoir associated with a water treatment plant (WTP), which provides the city of Murcia (Spain) with drinking water. Water samples were collected in different stages of the treatment. In the reservoir, the presence of dissolved and intracellular microcystins is constant, both in benthos and in plankton. The collected samples show a positive correlation between the dissolved microcystins and the benthic ones in the reservoir itself, as well as in an upstream reservoir (Ojós Reservoir). The treatment process (ozone+clarification+ozone+activated carbon) is very effective in the removal of toxins, and the drinking water produced is totally free of microcystins. The incorporation of the benthic communities in the routine check for the presence of microcystins is recommended, since it is not compulsory according to the current legislation.

In 1998, the protected area of Doñana, an important natural region in SW Europe, was affected with great amount of acidic waters and sludge from a pyrite mine loaded with toxic metals such as thallium (Tl). Since this ecological catastrophe, several studies have addressed the effects of this pollution on the flora and fauna in this protected area. However, in contrast to other non-essential metals, scarce information on Tl was available after this disaster, especially in terrestrial environments. This study reported a 3- and 10-fold increase in Tl in liver and kidneys, respectively, of the greater white-toothed shrew, Crocidura russula, in the polluted site in comparison with reference animals. Kidneys showed the highest concentrations of this metal in the polluted site, whereas both organs analysed have similar concentrations in the reference site. Although no significant age-dependent variation was found, adults had higher concentrations than juveniles. Moreover, females showed higher concentrations than males. These results demonstrate the high entrance and transfer of Tl in terrestrial food-chains. To the best of my knowledge, these data constitute the first measurements of Tl in mammals from the protected area of Doñana and are among the few available for insectivorous mammals.

The aims of the study were to evaluate toxic and essential metal concentrations in meat and offal from pigs in north-west Spain to compare these with reported metal concentrations in pigs in other countries and in cattle in this region, and to relate the observed concentrations to maximum acceptable concentrations. Samples from 63 pigs aged 6 months were randomly collected at slaughter. After acid digestion, levels of metals were determined by ICP-OES and ICP-MS. As regards the toxic metals, mean concentrations in liver, kidney and muscle were 0.073, 0.308 and 0.009 mg kg(-1) fresh weight for cadmium, 0.004, 0.008 and 0.003 mg kg(-1) for lead, 0.013, 0.011 and 0.003 mg kg(-1) for arsenic, and 0.001, 0.002 and 0.001 mg kg(-1) for mercury. These concentrations can be considered low, and in general similar to those reported in similar studies in recent years. In addition, maximum admissible concentrations established by the European Union were not exceeded in any sample. As regards the essential metals, concentrations in liver, kidney and muscle were 14.9, 5.63 and 6.85 mg kg(-1) for copper, 81.3, 28.9 and 42.5 mg kg(-1) for zinc, 195, 51.6 and 26.5 mg kg(-1) for iron; 1.17, 2.51 and 0.656 mg kg(-1) for selenium, 3.32, 1.56 and 1.01 mg kg(-1) for manganese, 0.023, 0.027 and 0.003 mg kg(-1) for cobalt, 0.120, 0.077 and 0.131 mg kg(-1) for chromium, 0.009, 0.027 and 0.026 mg kg(-1) for nickel, and 1.62, 0.683 and 0.140 mg kg(-1) for molybdenum. These concentrations are all within the accepted adequate-safe ranges for this animal species, and in general are in line with those previously reported in the literature.

Barbel (Barbus graellsii) is a freshwater fish used as a sentinel species in environmental monitoring programmes and ecotoxicological studies in northern Spain, particularly in the Ebro River basin, the largest freshwater resource in Spain. We developed specific primers for the quantification of CYP1A and metallothionein (MT)-1 and -2 gene expression by QRT-PCR in barbel in order to assess their suitability in biological effect monitoring of dioxin-like compounds and metals. All three genes responded as expected in laboratory tests, using model inducers. Hepatic CYP1A mRNA levels showed a twofold induction in fish injected intraperitoneally with beta-naphthoflavone, whereas MT-1 and MT-2 gene expression was strongly induced by cadmium (15- and 13-fold, respectively) and mercury (five- and eightfold). Barbel populations from different sites on the Ebro River basin showed a good correlation between the historical records of organochlorine pollution, CYP1A expression levels and EROD activity. Nevertheless, although metallothionein protein levels in the liver of wild fish correlated with hepatic levels of mercury, MT-1 and MT-2 gene expression did not correlate with the mercury content or with the concentration of metals in sediments from the sites inhabited by the fish. These results demonstrate the utility of barbel CYP1A-mRNA expression, but not that of MT-1 or MT-2, as a biomarker in field studies. The tools and protocols developed here are likely to apply to other species of the genus Barbus, with some 700 species distributed throughout most of the Old World.

Twenty-three barbels (Barbus graellsii), 30 bleaks (Alburnus alburnus) and 4 sediment samples were collected from four areas of the Cinca River (NE Spain), upstream and downstream of a mercury cell chlor-alkali plant. Mercury concentration in sediments downstream of the plant was about 25 times higher than the concentration upstream. Mercury levels in the muscle and liver of barbels downstream of the chlor-alkali plant were 10 and 30 times higher than those upstream. The average mercury concentration in the muscle and liver of barbels downstream of the factory was 1.48 and 1.78mg/kgw.w., with maximal concentrations of 2.43 and 3.64mg/kgw.w, respectively. Liver/muscle ratio of mercury was significantly higher in barbels sampled downstream of the factory. Bleak showed a similar pattern, with a significantly higher whole-body mercury concentration downstream of the chlor-alkali plant. Nevertheless, a progressive decrease in mercury concentration downstream of the impact area was found in bleak, but not in barbel. Histopathological analysis showed that fish downstream of the factory had significantly higher prevalence of liver pathologies (p<0.05). Although there are no specific histopathological markers of mercury exposure, barbels with the highest Hg levels in the liver also showed the most severe pathologies, and the processes found in these barbels were consistent with the pro-oxidant effect of heavy metals.

The aim of the present study was to assess whether the use of pig slurry and associated copper accumulation in calves grazing pastures fertilized with pig slurry from the Deza region in NW Spain has a significant influence on toxic metal and essential metal levels, and to investigate whether copper accumulation is correlated with the levels of these other metals. Correlations between copper, toxic metals (cadmium, lead) and essential metals (molybdenum, iron, zinc, selenium, manganese and cobalt) concentrations were evaluated in liver and kidney of 195 calves from the region of Deza. Metal concentrations were determined by ICP-OES and correlations between pairs of elements by Pearson correlation analysis. There was a strongly significant positive association between copper and lead in the liver. Considering the trace metals, copper in the liver showed significant positive correlations with cobalt in both liver and kidney, and with molybdenum in the liver. In addition, significant correlations were observed between copper and most essential metals in kidney. These associations are similar to those found in different animal species experimentally dosed with copper and other metals, as well as in cattle exposed to normal copper concentrations.

Sand gobies, Pomatoschistus minutus, were collected monthly from September 2002 to August 2003 at a station situated 8 km upstream from the mouth of the Guadalquivir estuary (southwest Spain). Physical parameters of the water and selected biomarkers of organic pollution were recorded in the fish to discuss its potential as a sentinel species in estuaries. The biomarkers selected were the activities of catalase (CAT), 7-ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST) in the liver and acetylcholinesterase (AChE) and lipid peroxidation (LP) in the head. The results showed an increase in total protein synthesis in late spring and early summer coinciding with the reproductive period as well as the release of fresh water from a dam situated 110 km upstream. During the same period, a significant depletion of hepatic GST and head AChE but higher LP levels in this tissue suggest exposure to pesticides such as those applied to crops established along the course of the river and reaching the estuary mostly when the freshwater discharges occur. Changes in CAT and EROD activities fluctuated randomly and were not noted as seasonally dependent. Biomarker fluctuations in sand goby are discussed as normal seasonal variations, but other variables-such as potential local pollution inputs-cannot be disregarded.

Liver samples of 42 birds belonging to 6 species of Ciconiiformes (grey heron (n = 17), little egret (n = 12), cattle egret (n = 1), glossy ibis (n = 1), little bittern (n = 1), and white stork (n = 10)), collected from two different zones of Spain (Ebro Delta and Madrid) in 1992-1997, were analyzed for organochlorine pesticides and PCBs. SigmaPCBs (sum of concentrations of individual congeners), p,p'-DDE, HCB, gamma-HCH, and heptachlor epoxide were the most prevalent residues detected in all samples (occurrence > 95%). There were no statistically significant differences in organochlorine levels between grey herons and little egrets, while levels of SigmaPCBs, p,p'-DDE, HCB, gamma-HCH, and heptachlor were significantly lower for white storks. These results could be explained by the different habitat of these species, aquatic in the Ebro Delta (grey herons, little egrets) and dry in Madrid (white stork), and their diverse feeding habits.

The intracellular distribution of copper (Cu) and zinc (Zn) in the livers of normal and moderately Cu-exposed cattle was investigated with the aim of improving understanding of the pathophysiology of Cu accumulation in cattle. At total liver Cu concentrations within the generally accepted normal range (25-100 mg/kg fresh weight) the large-granule fraction was the main subcellular compartment for Cu accumulation, followed by the cytosol and the nucleus, whereas in the microsomal fraction Cu accumulation was very low. With increasing Cu exposure, the capacity of the large-granule fraction to accumulate Cu decreased, proceeding towards a plateau (estimated at about 80 mg/kg at a projected total liver Cu concentrations of 450 mg/kg), accompanied by progressively greater Cu accumulation in the nucleus and cytosol. Total liver Cu concentration had little influence on subcellular Zn distribution, with hepatic Cu concentrations being only moderately correlated with Zn concentration in the large-granule fraction. There was a strong association between the proportion of total Cu bound to metallothionein (MT) and the proportion of total Zn in the large-granule fraction, suggesting that Zn displaced from MT is taken up by the lysosomes for excretion. This pattern of Cu accumulation, as in sheep, may be due to the limited capacity for metallothionein binding of Cu and excretion in bile.