Benjamin Piña

Detection and evaluation of endocrine-disruption activity in water samples from Portuguese rivers

Water samples (n = 183) from Portuguese rivers were tested for the presence of endocrine disruptors using the recombinant yeast assay (RYA) combined with chemical identification of compounds having endocrine-disruption properties by liquid chromatography coupled to mass spectrometry. Ten selected locations were sampled monthly for a period of 20 months, from April 2001 to December 2002. More than 90% of samples showed either no detectable or low levels of estrogenicity (<0.1 ng/L of estradiol equivalents). The remaining samples (17 in total, 9.3%) showed estrogenicity values ranging from 0.1 to 1.7 ng/L of estradiol equivalents; only two samples showed values greater than 1 ng/L of estradiol equivalents. Most highly estrogenic samples (13 of 17 samples) originated in five sampling sites clustered in two zones near Porto and Lisbon. Chemical analysis detected alkylphenolic compounds (octyl- and nonylphenol plus nonylphenol ethoxylates) in all samples, albeit at concentrations less than 1 microg/L for each compound in 80% of samples. Total analyte concentration exceeded 10 microg/L in only 10 samples, with all but one of those originating from only two sampling sites. In these two locations, a good correlation was observed between the concentrations of octylphenol, nonylphenol, and to a lesser extent, bisphenol A in the samples and their estrogenicity values as calculated by RYA. We conclude that estrogenic activity can be explained by alkylphenol contamination in only these sites; for the remainder, we propose that pesticides and urban waste may be the main factors responsible for estrogenic contamination.

DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy

The centromeric dodeca‐satellite of Drosophila forms altered DNA structures in vitro in which its purine‐rich strand (G‐strand) forms stable fold‐back structures, while the complementary C‐strand remains unstructured. In this paper, the purification and characterization of DDP1, a single‐stranded DNA‐binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca‐satellite C‐strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca‐satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co‐localizing with the heterochromatin protein HP1. DDP1 is a multi‐KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Δscp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.

Characterization of the multixenobiotic resistance (MXR) mechanism in embryos and larvae of the zebra mussel (Dreissena polymorpha) and studies on its role in tolerance to single and mixture combinations of toxicants

The study of the cellular mechanisms of tolerance of organisms to pollution is a key issue in aquatic environmental risk assessment. Recent evidence indicates that multixenobiotic resistance (MXR) mechanisms represent a general biological defense of many marine and freshwater organisms against environmental toxicants. In this work, toxicologically relevant xenobiotic efflux transporters were studied in early life stages of zebra mussels (Dreissena polymorpha). Expression of a P-gp1 (ABCB1) transporter gene and its associated efflux activities during development were studied, using qRT-PCR and the fluorescent transporter substrates rhodamine B and calcein-AM combined with specific transporter inhibitors (chemosensitizers). Toxicity bioassays with the model P-gp1 chemotherapeutic drug vinblastine applied singly and in combination with different chemosensitizers were performed to elucidate the tolerance role of the P-gp1 efflux transporter. Results evidenced that the gene expression and associated efflux activities of ABC transporters were low or absent in eggs and increased significantly in 1-3d old trochophora and veliger larvae. Specific inhibitors of Pgp1 and/or MRP transport activities including cyclosporine A, MK571, verapamil and reversin 205 and the musk celestolide resulted in a concentration dependent inhibition of related transport activities in zebra mussel veliger larvae, with IC50 values in the lower micromolar range and similar to those reported for mammals, fish and mussels. Binary mixtures of the tested transporter inhibitors except celestolide with the anticancer drug and P-gp1 substrate vinblastine increased the toxicity of the former compound more than additively. These results indicate that MXR transporter activity is high in early life-stages of the zebra mussel and that may play an important role in the tolerance to environmental contaminants.

Physiological responses to mercury in feral carp populations inhabiting the low Ebro River (NE Spain), a historically contaminated site

The low Ebro River course (Northeast Spain) is historically affected by mercury pollution due to a chlor-alkali plant operating at the town of Flix for more than a century. River sediments analysed during the last 10 years showed high mercury levels in the river section starting just downstream the factory and spanning some 90km, down to the river delta. The possible environmental impact was studied by a combination of field and laboratory studies. Mercury concentrations in liver, kidney and muscle of feral carp (Cyprinus carpio) sampled downstream Flix were one to two orders of magnitude higher than those from carps sampled upstream Flix. Elevated levels of mercury in these samples associated with significant increases on the concentration of reduced glutathione (GSH) in liver and on mRNA expression of two metallothionein genes, MT1 and MT2, in kidney and, partially, in scales, but not in liver. Conversely, no biochemical evidence for oxidative stress or DNA damage was found in these tissues. Non-contaminated carps subjected to intraperitoneal mercury injection resulted in a 20-fold increase of MT1 and MT2 mRNA levels in carp kidney, with minimal changes in liver levels. Our data suggests the coordinate increase of metallothionein mRNA in kidney and of GSH in liver constitutes an excellent marker of exposure to sub-toxic mercury levels in carps. This study also demonstrates that apparently healthy fish populations may exceed the mercury contamination acceptable for human consumption.

Ecological relevance of biomarkers in monitoring studies of macro-invertebrates and fish in Mediterranean rivers.

Mediterranean rivers are probably one of the most singular and endangered ecosystems worldwide due to the presence of many endemic species and a long history of anthropogenic impacts. Besides a conservation value per se, biodiversity is related to the services that ecosystems provide to society and the ability of these to cope with stressors, including climate change. Using macro-invertebrates and fish as sentinel organisms, this overview presents a synthesis of the state of the art in the application of biomarkers (stress and enzymatic responses, endocrine disruptors, trophic tracers, energy and bile metabolites, genotoxic indicators, histopathological and behavioural alterations, and genetic and cutting edge omic markers) to determine the causes and effects of anthropogenic stressors on the biodiversity of European Mediterranean rivers. We also discuss how a careful selection of sentinel species according to their ecological traits and the food-web structure of Mediterranean rivers could increase the ecological relevance of biomarker responses. Further, we provide suggestions to better harmonise ecological realism with experimental design in biomarker studies, including statistical analyses, which may also deliver a more comprehensible message to managers and policy makers. By keeping on the safe side the health status of populations of multiple-species in a community, we advocate to increase the resilience of fluvial ecosystems to face present and forecasted stressors. In conclusion, this review provides evidence that multi-biomarker approaches detect early signs of impairment in populations, and supports their incorporation in the standardised procedures of the Water Frame Work Directive to better appraise the status of European water bodies.

Evaluation of fungal- and photo-degradation as potential treatments for the removal of sunscreens BP3 and BP1.

Photodecomposition might be regarded as one of the most important abiotic factors affecting the fate of UV absorbing compounds in the environment and photocatalysis has been suggested as an effective method to degrade organic pollutants. However, UV filters transformation appears to be a complex process, barely addressed to date. The white rot fungus Trametes versicolor is considered as a promising alternative to conventional aerobic bacterial degradation, as it is able to metabolise a wide range of xenobiotics. This study focused on both degradation processes of two widely used UV filters, benzophenone-3 (BP3) and benzophenone-1 (BP1). Fungal treatment resulted in the degradation of more than 99% for both sunscreens in less than 24 h, whereas photodegradation was very inefficient, especially for BP3, which remained unaltered upon 24 h of simulated sunlight irradiation. Analysis of metabolic compounds generated showed BP1 as a minor by-product of BP3 degradation by T. versicolor while the main intermediate metabolites were glycoconjugate derivatives. BP1 and BP3 showed a weak, but significant estrogenic activity (EC50 values of 0.058 mg/L and 12.5 mg/L, respectively) when tested by recombinant yeast assay (RYA), being BP1 200-folds more estrogenic than BP3. Estrogenic activity was eliminated during T. versicolor degradation of both compounds, showing that none of the resulting metabolites possessed significant estrogenic activity at the concentrations produced. These results demonstrate the suitability of this method to degrade both sunscreen agents and to eliminate estrogenic activity.

A genomic and ecotoxicological perspective of DNA array studies in aquatic environmental risk assessment

Ecotoxicogenomics is developing into a key tool for the assessment of environmental impacts and environmental risk assessment for aquatic ecosystems. This review aims to report achievements and drawbacks of this technique and to explore potential conceptual and experimental procedures to improve future investigations. Ecotoxicogenomic literature evidences the ability of genomic technologies to characterize toxicant specific gene transcriptome patterns that can be used to identify major toxicants affecting aquatic species. They also contribute decisively to the development of new molecular biomarkers and, in many cases, to the determination of new possible gene targets. Primary transcriptomic responses obtained after short exposures provided more information of putative gene targets than secondary responses obtained after long, chronic exposures, which in turn are usually more accurate to describe actual environmental impacts in natural populations. Several problems need to be addressed in future investigations: the lack of studies (and genomic information) on key ecological species and taxa, the need to better understand the different transcriptomic responses to high and low doses and, especially, short and long exposures, and the need to improve experimental designs to minimize false transcriptome interpretations of target genes.

Identification of water soluble and particle bound compounds causing sublethal toxic effects. A field study on sediments affected by a chlor-alkali industry

A combination of cost effective sublethal Daphnia magna feeding tests, yeast- and cell culture-based bioassays and Toxicity Identification Evaluation (TIE) procedures was used to characterize toxic compounds within sediments collected in a river area under the influence of the effluents from a chlor-alkali industry (Ebro River, NE Spain). Tests were designed to measure and identify toxic compounds in the particulate and filtered water fractions of sediment elutriates. The combined use of bioassays responding to elutriates and dioxin-like compounds evidenced the existence of three major groups of hazardous contaminants in the most contaminated site: (A) metals such as cadmium and mercury bound to sediment fine particles that could be easily resuspended and moved downstream, (B) soluble compounds (presumably, lye) able to alkalinize water to toxic levels, and (C) organochlorine compounds with high dioxin-like activity. These results provided evidence that elutriate D. magna feeding responses can be used as surrogate assays for more tedious chronic whole sediment tests, and that the incorporation of such tests in sediment TIE procedures may improve the ability to identify the toxicity of particle-bound and water-soluble contaminants in sediments.

Topoisomerase II is required for the production of long Pol II gene transcripts in yeast

The extent to which the DNA relaxation activities of eukaryotic topoisomerases (topo I and topo II) are redundant during gene transcription is unclear. Although both enzymes can often substitute for each other in vivo, studies in vitro had revealed that the DNA cross-inversion mechanism of topo II relaxes chromatin more efficiently than the DNA strand-rotation mechanism of topo I. Here, we show that the inactivation of topo II in budding yeast produces an abrupt decrease of virtually all polyA+ RNA transcripts of length above ∼3 kb, irrespective of their function. This reduction is not related to transcription initiation but to the stall of RNA polymerase II (Pol II) during elongation. This reduction does not occur in topo I mutants; and it is not avoided by overproducing yeast topo I or bacterial topo I, which relaxes (−) DNA supercoils. It is rescued by catalytically active topo II or a GyrBA enzyme, which relaxes (+) DNA supercoils. These findings demonstrate that DNA relaxation activities of topo I and topo II are not interchangeable in vivo. Apparently, only topo II relaxes efficiently the (+) DNA supercoils that stall the advancement of Pol II in long genes. A mechanistic model is proposed.

Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults.

Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations.