Joana Lourenço

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC50=145.7 mg/L) than adults (96 h-LC50=39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant.

Genotoxic endpoints in the earthworms sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides

Eisenia andrei was exposed, for 56 days, to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure, to assess metals bioaccumulation, coelomocytes DNA integrity and cytotoxicity. Radionuclides bioaccumulation and growth were also determined at 0 h, 14 and 56 days of exposure. Results have shown the bioaccumulation of metals and radionuclides, as well as, growth reduction, DNA damages and cytotoxicity in earthworms exposed to contaminated soil. The usefulness of the comet assay and flow cytometry, to evaluate the toxicity of contaminants such as metals and radionuclides in earthworms are herein reported. We also demonstrated that DNA strand breakage and immune cells frequency are important endpoints to be employed in the earthworm reproduction assay, for the evaluation of soil geno and cytotoxicity, as part of the risk assessment of contaminated areas. This is the first study that integrates DNA damage and cytotoxicity evaluation, growth and bioaccumulation of metals and radionuclides in a sub lethal assay, for earthworms exposed to soil contaminated with metals and radionuclides.

The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives

Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers. (iv) To build a constructive criticism and summarize the needs for protocol improvements for future test applications within the field of ecotoxicology. The Comet Assay is still developing and its potential is yet underexploited in experimental models, mesocosmos or natural ecosystems.

Evaluation of the sensitivity of genotoxicity and cytotoxicity endpoints in earthworms exposed in situ to uranium mining wastes

Earthworms were exposed for 56 days to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The exposure occurred in situ: the containers with contaminated soil were placed near the mine pit; the containers with reference soil were placed in a reference site. For the assessment of metals bioaccumulation, DNA damages, cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation, organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure. For the assessment of radionuclides bioaccumulation, animals were sampled after 0, 14 and 56 days of exposure. As for growth, organisms were sampled after 0, 14, 28 and 56 days of exposure. The reproduction assay was performed according to the OECD (2004) guideline. DNA damages were assessed by comet assay and flow cytometry was used to determine cell-to-cell variation in DNA content, Median Fluorescence Intensity (MFI), coelomocytes frequency and proliferation. Results have shown a myriad of effects in the organisms exposed to the contaminated soil, namely: the inhibition of reproduction, growth reduction, DNA damages, cytotoxicity, changes in eleocytes fluorescence intensity, coelomocytes proliferation and bioaccumulation of metals and radionuclides. Our results showed that the evaluation of genotoxicity and cytotoxicity endpoints, along with other parameters at an individual level in standard reproduction assays conducted in situ, are important to improve the risk assessment process of areas contaminated with uranium and other radioactive mining wastes.

Histopathological changes in the earthworm Eisenia andrei associated with the exposure to metals and radionuclides

Earthworms were exposed for 56 d to a contaminated soil, from an abandoned uranium mine, and to the natural reference soil LUFA 2.2. Histological changes in earthworms body wall (epidermis, circular and longitudinal muscles) and gastrointestinal tract (chloragogenous tissue and intestinal epithelium) were assessed, after 0, 14 and 56 d of exposure. Results have shown alterations in all the studied tissues after 14 d of exposure (except for the intestinal epithelium), yet more severe effects were registered after 56 d of exposure. Herein we report histopathological alterations as a good biomarker for the evaluation of soil quality. We also demonstrate that morphological changes in the body wall and gastrointestinal tract, are important endpoints that could be added to earthworms standardized tests, for the evaluation of soil toxicity, as part of the risk assessment of contaminated areas.

Ecotoxicity and genotoxicity of a binary combination of triclosan and carbendazim to Daphnia magna

In the environment, chemical substances appear as complex mixtures and consequently organisms are exposed to a variety of chemicals from different sources (e.g. wastewater treatment plants, agriculture runoffs). When studying chemical mixtures, there are two conceptual models usually used to predict toxicity: the Independent Action (IA) and Concentration Addition (CA) models. However, deviations from these reference models can occur as synergism or antagonism, dose ratio or dose level dependency. The aim of the present study was to investigate the effects of triclosan and carbendazim, and their binary mixture to Daphnia magna. With this purpose, immobilisation, feeding inhibition, and reproduction were assessed as main ecotoxicity endpoints. In addition, in vivo genotoxicity of both chemicals was investigated using the comet assay. In the single exposure, carbendazim was more toxic to D. magna than triclosan. When daphnids were exposed to both single compounds, DNA damage was observed. Concerning mixture exposures, different endpoints followed different patterns of response, from additivity: IA model (feeding inhibition and reproduction data), to deviations that indicate interaction between chemicals inside the organism: dose level dependency (immobilisation data) and dose ratio dependency (DNA damage). This study showed that additivity does not rule the dose-effect relation in chemical mixtures of carbendazim and triclosan and interactions between both chemicals might induce generally higher toxicity than predicted based on single chemical exposures.

Biomonitoring a human population inhabiting nearby a deactivated uranium mine.

Environmental exposure to uranium and its daughter radionuclides, has been linked to several negative effects such as those related with important physiological processes, like hematopoiesis, and may also be associated with genotoxicity effects. Herein, genotoxic effects, immunotoxicity, trace elements and C reactive protein (CRP) analyses, were performed in peripheral blood samples collected from individuals of a population living near a deactivated uranium mine. C reactive protein analysis was performed to exclude candidates with active inflammatory processes from further evaluations. DNA damage and immunotoxicity (immunophenotyping and immune cell counts) were evaluated by comet assay and flow cytometry, respectively. Significant DNA damage was observed in the peripheral blood samples from volunteers living in the Cunha Baixa village. A significant decrease of NK and T lymphocytes counts were observed in the individuals from the Cunha Baixa village, when compared with individuals from the reference site. Uranium and manganese levels were significantly higher in the Cunha Baixa village inhabitants. On the other hand, zinc levels were significantly lower in those individuals when compared with the volunteers from the control village. Results suggest that inhabitants from Cunha Baixa have a higher risk of suffering from serious diseases such as cancer, since high DNA damages were observed in peripheral blood leukocytes and also decreased levels of NK and T cells, which play an essential role in the defense against tumor growth.

Metal bioaccumulation, genotoxicity and gene expression in the European wood mouse (Apodemus sylvaticus) inhabiting an abandoned uranium mining area.

Genotoxic effects caused by the exposure to wastes containing metals and radionuclides were investigated in the European wood mice (Apodemus sylvaticus). The animals were captured in the surroundings of an abandoned uranium mining site. DNA damage was assessed by comet assay; gene expression and single nucleotide polymorphisms (SNPs) were assessed, respectively, by Real-Time PCR and melt curve analysis. The bioaccumulation of metals in the liver, kidney and bones was also determined to help clarify cause-effect relationships. Results confirmed the bioaccumulation of cadmium and uranium in organisms exposed to uranium mining wastes. P53 gene was found to be significantly up-regulated in the liver of those organisms and SNPs in the Rb gene were also detected in the kidney. Our results showed that uranium mining wastes caused serious DNA damage resulting in genomic instability, disclosed by the significant increase in DNA strand breaks and P53 gene expression disturbance. These effects can have severe consequences, since they may contribute for the emergence of serious genetic diseases. The fact that mice are often used as bioindicator species for the evaluation of risks of environmental exposure to humans, raises concerns on the risks for human populations living near uranium mining areas.

Transcript profiling and DNA damage in the European eel (Anguilla anguilla L.) exposed to 7,12-dimethylbenz[a]anthracene

The molecular responses induced during and after an acute exposure to 7,12-dimethylbenz[a]anthracene (DMBA) were analysed in liver, gill and blood cells of juvenile Anguilla anguilla with the aim of developing molecular biomarkers of environmental PAH pollution. Changes in the mRNA expression levels of the cell cycle checkpoint-related rad1 gene and the mRNAs of differentially expressed genes by suppression subtractive hybridization (SSH) were analysed in the liver, and related to well-established biomarkers: cyp1A1 mRNA expression and assessment of the DNA integrity using the comet assay and flow cytometry. DMBA exposure resulted in increased cyp1A1 mRNA levels, suggesting that cyp1A1 might be involved in the metabolism of DMBA. Global DNA damage, detected by the comet assay, was observed in the three tissues analysed but only blood cells showed chromosomal lesions as analysed by flow cytometry. Although DNA damage was found in the liver, no induction in rad1 gene was observed in this organ. The global SSH approach revealed that mRNAs of genes related to xenobiotic metabolism, immune processes and cytoskeleton dynamics were differentially expressed in DMBA-exposed eel livers, highlighting the complexity in the response observed in fish exposed to a genotoxic agent and providing directions for new biomarker development.

SSH gene expression profile of Eisenia andrei exposed in situ to a naturally contaminated soil from an abandoned uranium mine

The effects of the exposure of earthworms (Eisenia andrei) to contaminated soil from an abandoned uranium mine, were assessed through gene expression profile evaluation by Suppression Subtractive Hybridization (SSH). Organisms were exposed in situ for 56 days, in containers placed both in a contaminated and in a non-contaminated site (reference). Organisms were sampled after 14 and 56 days of exposure. Results showed that the main physiological functions affected by the exposure to metals and radionuclides were: metabolism, oxireductase activity, redox homeostasis and response to chemical stimulus and stress. The relative expression of NADH dehydrogenase subunit 1 and elongation factor 1 alpha was also affected, since the genes encoding these enzymes were significantly up and down-regulated, after 14 and 56 days of exposure, respectively. Also, an EST with homology for SET oncogene was found to be up-regulated. To the best of our knowledge, this is the first time that this gene was identified in earthworms and thus, further studies are required, to clarify its involvement in the toxicity of metals and radionuclides. Considering the results herein presented, gene expression profiling proved to be a very useful tool to detect earthworms underlying responses to metals and radionuclides exposure, pointing out for the detection and development of potential new biomarkers.