ABSTRACT
Pesticides in the environment often compromise the ecosystem, thus requiring reliable approaches to assess their effects. Commonly used approaches, such as in vivo, come with several disadvantages, namely in the light of the 3 R's policy. Seeking for accurate and ethical approaches, this study intended to validate the ex vivo technique as an alternative, and to assess the genotoxicity of chemically-based pesticides and a biopesticide. The ex vivo approach was applied to gill cells of Procambarus clarkii for 2, 4 and 8 h. Cell viability and DNA integrity were evaluated to determine the applicability of this approach. Crayfish gill cells only showed to be suitable for exposures of 2 h. Accordingly, genotoxicity was evaluated in gill cells exposed, for 2 h, to environmentally relevant concentrations of the chemically-based pesticides dimethoate (20 µg L-1), imazalil (160 µg L-1) and penoxsulam (23 µg L-1), as well as to the bioinsecticide Turex® (25, 50, 100, 200 and 400 µg L-1). Every chemically-based pesticide demonstrated to be genotoxic, despite not inducing oxidative DNA damage. On the other hand, Turex® showed no genotoxic effects. Overall, the ex vivo approach demonstrated to be possible and practical to implement, improving the number of outcomes with a lower number of organisms. The findings from the screening test suggest that biological pesticides may pose a lower risk to non-target organisms compared to chemically-based pesticides.
Subject(s)
Astacoidea , DNA Damage , Gills , Pesticides , Animals , Pesticides/toxicity , Gills/drug effects , DNA Damage/drug effects , Astacoidea/drug effects , Risk Assessment , Mutagenicity Tests , Water Pollutants, Chemical/toxicity , Cell Survival/drug effects , Comet Assay , Mutagens/toxicityABSTRACT
Population growth leads to the need for more efficient techniques and compounds in agriculture, such as pesticides, to deal with the ever-growing demand. Pesticides may end up in the environment, often compromising the ecosystem affecting all organisms including humans. Therefore, the consequences of exposure to these compounds to biota and humans needs to be assessed. Bearing this in mind, the aim of this study was to examine the in vitro cytotoxicity and genotoxicity attributed to exposure to the biopesticide Turex® utilizing the liver cell line HepG2. Cells were incubated with biopesticide Turex® at 250, 500, 1000, 1500 or 2000 µg/L in both non-activated and activated forms for 24 and 48 h. Subsequent effects on cell viability were assessed using the MTT. The influence on cell cycle dynamics was determined by flow cytometry, while DNA damage was measured by the comet assay. Data demonstrated that activated Turex® induced cytotoxicity and DNA damage after 48 h in HepG2 cell line. The cell cycle progression was not markedly affected by Turex® at any concentration or duration of exposure. In conclusion, data demonstrated the potential adverse effects attributed to exposure to biopesticide Turex® in human cell line HepG2. Consequently, this type of biopesticide needs to be further investigated to determine the potential adverse in vivo effects on various non-target organisms.
Subject(s)
Biological Control Agents , Pesticides , Humans , Hep G2 Cells , Biological Control Agents/pharmacology , Ecosystem , DNA Damage , Cell Cycle Checkpoints , Pesticides/toxicity , Cell Cycle , Cell SurvivalABSTRACT
The present study aimed to evaluate the reprotoxicity of environmental (0.25 µg.L-1) and supra-environmental (25 µg.L-1 and 250 µg.L-1) levels of silver nanoparticles (Ag NP) on the Pacific oyster (Magallana gigas), by determining sperm quality. For that, we evaluated sperm motility, mitochondrial function and oxidative stress. To determine whether the Ag toxicity was related to the NP or its dissociation into Ag ions (Ag+), we tested the same concentrations of Ag+. We observed no dose-dependent responses for Ag NP and Ag+, and both impaired sperm motility indistinctly without affecting mitochondrial function or inducing membrane damage. We hypothesize that the toxicity of Ag NP is mainly due to adhesion to the sperm membrane. Blockade of membrane ion channels may also be a mechanism by which Ag NP and Ag+ induce toxicity. The presence of Ag in the marine ecosystem is of environmental concern as it may affect reproduction in oysters.
Subject(s)
Metal Nanoparticles , Ostreidae , Male , Animals , Metal Nanoparticles/toxicity , Silver/toxicity , Ecosystem , Sperm Motility , Semen , IonsABSTRACT
Titanium dioxide nanoparticles (TiO2 NP) were reported to be reprotoxic in humans and fish. However, the effects of these NP on the reproduction of marine bivalves, namely oysters, remain unknown. Thus, a short-term (1â¯h) direct exposure of sperm of the Pacific oyster (Crassostrea gigas) to two TiO2 NP concentrations (1 and 10â¯mg.L-1) was performed, and sperm motility, antioxidant responses, and DNA integrity were evaluated. Although no changes occurred in sperm motility and the activities of the antioxidants, the genetic damage indicator increased at both concentrations, demonstrating that TiO2 NP affects the DNA integrity of oyster sperm. Although DNA transfer can happen, it does not fulfill its biological mission since the transferred DNA is not intact and may compromise the reproduction and recruitment of the oysters. This vulnerability of C. gigas sperm towards TiO2 NP highlights the importance of studying the effects of NPs exposure to broadcast spawners.
Subject(s)
Crassostrea , Nanoparticles , Water Pollutants, Chemical , Animals , Humans , Male , Water Pollutants, Chemical/toxicity , Sperm Motility , Semen , Spermatozoa , DNA , Antioxidants/pharmacologyABSTRACT
The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.
Subject(s)
DNA Damage , Pyrimidine Dimers , Animals , Humans , Comet Assay/methods , Eukaryotic Cells , DNA/geneticsABSTRACT
The identification of novel feed materials as a source of functional ingredients is a topical priority in the finfish aquaculture sector. Due to the agrotechnical practices associated and phytochemical profiling, halophytes emerge as a new source of feedstuff for aquafeeds, with the potential to boost productivity and environmental sustainability. Therefore, the present study aimed to assess the potential of Salicornia ramosissima incorporation (2.5, 5, and 10%), for 2 months, in the diet of juvenile European seabass, seeking antioxidant (in the liver, gills, and blood) and genoprotective (DNA and chromosomal integrity in blood) benefits. Halophyte inclusion showed no impairments on growth performance. Moreover, a tissue-specific antioxidant improvement was apparent, namely through the GSH-related defense subsystem, but revealing multiple and complex mechanisms. A genotoxic trigger (regarded as a pro-genoprotective mechanism) was identified in the first month of supplementation. A clear protection of DNA integrity was detected in the second month, for all the supplementation levels (and the most prominent melioration at 10%). Overall, these results pointed out a functionality of S. ramosissima-supplemented diets and a promising way to improve aquaculture practices, also unraveling a complementary novel, low-value raw material, and a path to its valorization.
ABSTRACT
The harmful effects of pesticides can be extended beyond the exposure time scale. Appraisals combining exposure and long-term post-exposure periods appear as an unavoidable approach in pesticide risk assessment, thus allowing a better understanding of the real impact of agrochemicals in non-target organisms. This study aimed to evaluate the progression of genetic damage in somatic and germ tissues of the crayfish Procambarus clarkii, also seeking for gender-specificities, following exposure (7 days) to penoxsulam (23 µg L-1 ) and a post-exposure (70 days) period. The same approach was applied to the model genotoxicant ethyl methanesulfonate (EMS; 5 mg L-1 ) as a complementary mean to improve knowledge on genotoxicity dynamics (induction vs. recovery). Penoxsulam induced DNA damage in all tested tissues, disclosing tissue- and gender-specificities, where females showed to be more vulnerable than males in the gills, while males demonstrated higher susceptibility in what concerns internal organs, that is, hepatopancreas and gonad. Crayfish were unable to recover from the DNA damage induced by EMS in gills and hepatopancreas (both genders) as well as in spermatozoa. The genotoxicity in the hepatopancreas was only perceptible in the post-exposure period. Oxidative DNA lesions were identified in hepatopancreas and spermatozoa of EMS-exposed crayfish. The spermatozoa proved to be the most vulnerable cell type. It became clear that the characterization of the genotoxic hazard of a given agent must integrate a complete set of information, addressing different types of DNA damage, tissue- and gender-specificities, as well as a long-term appraisal of temporal progression of damage.
Subject(s)
Pesticides , Water Pollutants, Chemical , Agrochemicals , Animals , Astacoidea/genetics , Astacoidea/metabolism , DNA Damage , Ethyl Methanesulfonate/toxicity , Female , Germ Cells , Male , Sulfonamides , Uridine/analogs & derivatives , Water Pollutants, Chemical/metabolism , Water Pollutants, Chemical/toxicityABSTRACT
Epigenome is susceptible to modulation by environmental pressures-namely, through alterations in global DNA methylation, impacting the organism condition and, ultimately, reverberating on the phenotype of the subsequent generations. Hence, an intergenerational study was conducted, aiming to clarify the influence of genotoxicants on global DNA methylation of the crayfish Procambarus clarkii. Two subsequent generations were exposed to the herbicide penoxsulam (Px; 23 µg·L-1) and to the genotoxicant model ethyl methanesulfonate (EMS; 5 mg·L-1). Px did not induce changes in DNA methylation of adult crayfish (F0). However, the hypomethylation occurring in unexposed F1 juveniles demonstrated that the history of exposure per se can modulate epigenome. In F1 descendants of the Px-exposed group, methylome (hypermethylated) was more affected in males than in females. EMS-induced hypomethylation in adult females (F0), also showed gender specificity. In addition, hypomethylation was also observed in the unexposed F1 crayfish, indicating an intergenerational epigenetic effect. The modulatory role of past exposure to penoxsulam or to EMS also showed a dependency on the crayfish developmental stage. Overall, this research revealed that indirect experiences (events occurring in a predecessor generation) can have an impact even greater than direct experiences (present events) on the epigenetic dynamics.
ABSTRACT
This study aims to assess and compare the kinetics (accumulation/elimination) of the marine biotoxins okadaic acid (OA) and dinophysistoxin-1 (DTX1), between native (Ruditapes decussatus) and invasive (Ruditapes philippinarum) clam species, and their genotoxic effects and DNA recover capacity after, exposure to toxic dinoflagellates Prorocentrum lima. Clams were fed with P. lima for 5 days and then to non-toxic algae (post-exposure) during other 5 days. Toxin concentrations determined in clams by LC-MS/MS were related with DNA damage and repair assessment through the comet and base excision repair (BER) assays, respectively. Differential accumulation patterns were observed between the invasive and native species. The invasive species consistently and progressively accumulated the toxins during the first 24 h of exposure, while the native clams showed drastic variations in the toxin accumulation. Nevertheless, at the end of a 5 days of exposure period, the native clams presented higher toxin concentrations, nearly reaching the legal regulatory limit for human consumption. In addition, native clams were vastly affected by OA and DTX1, presenting an increment in the DNA damage since the first day, with a correspondent increase in the repair activity. On the other hand, invasive clams were not affected by the dinoflagellate toxins, exhibiting only some signs of the challenge, namely an increase in the DNA repair mechanisms in the post-exposure period. Invasive clams R. philippinarum are better adapted to cope with harmful algal blooms and OA-group toxins than native species. These results may increase farming interest and may lead to new introductions of the invasive clams. In sympatry sites, exposure to OA-group toxins may unbalance clams species biomass and distribution as exposure to toxic dinoflagellates affects the native clams from cellular to a population level, representing a significant threat to development and maintenance of R. decussatus populations.
Subject(s)
Bivalvia , Harmful Algal Bloom , Animals , Chromatography, Liquid , DNA , Humans , Tandem Mass Spectrometry , ToxicokineticsABSTRACT
UV filters are potentially harmful to marine organisms. Given their worldwide dissemination and the scarcity of studies on marine fish, we evaluated the toxicity of an organic (oxybenzone) and an inorganic (titanium dioxide nanoparticles) UV filter, individually and in a binary mixture, in the turbot (Scophthalmus maximus). Fish were intraperitoneally injected and a multi-level assessment was carried out 3 and 7 days later. Oxybenzone and titanium dioxide nanoparticles induced mild effects on turbot, both isolated and in mixture. Neither oxidative stress (intestine, liver and kidney) nor neurotoxicity (brain) was found. However, liver metabolic function was altered after 7 days, suggesting the impairment of the aerobic metabolism. An increased motility rate in oxybenzone treatment was the only behavioural alteration (day 7). The intestine and liver were preferentially targeted, while kidney and brain were unaffected. Both infra- and supra-additive interactions were perceived, with a toxicodynamic nature, resulting either in favourable or unfavourable toxicological outcomes, which were markedly dependent on the organ, parameter and post-injection time. The combined exposure to the UV filters did not show a consistent increment in toxicity in comparison with the isolated exposures, which is an ecologically relevant finding providing key information towards the formulation of environmentally safe sunscreen products.
Subject(s)
Benzophenones/toxicity , Nanoparticles/adverse effects , Neurotoxicity Syndromes/pathology , Oxidative Stress , Sunscreening Agents/toxicity , Titanium/toxicity , Ultraviolet Rays/adverse effects , Animals , Behavior, Animal , Flatfishes/growth & development , Nanoparticles/chemistry , Neurotoxicity Syndromes/etiology , Water Pollutants, Chemical/toxicityABSTRACT
Commonly affected by changes in climate and environmental conditions, coastal areas are very dynamic environments where shellfish play an important ecological role. In this study, the oxidative stress and genotoxic responses of mussels (Mytilus galloprovincialis) exposed to paralytic shellfish toxin (PST) - producing dinoflagellates Gymnodinium catenatum were evaluated under i) current conditions (CC: 19 °C; pH 8.0), ii) warming (W: 24 °C; pH 8.0), iii) acidification (A:19 °C; pH 7.6) and iv) combined effect of warming and acidification (WA: 24 °C; pH 7.6). Mussels were fed with G. catenatum for 5 days, and to a non-toxic diet during the following 10 days. A battery of oxidative stress biomarkers and comet assay was performed at the peak of toxin accumulation and at the end of the post-exposure phase. Under CC, gills and hepatopancreas displayed different responses/vulnerabilities and mechanisms to cope with PST. While gills presented a tendency for lipid peroxidation (LPO) and genetic damage (expressed by the Genetic Damage Indicator - GDI), hepatopancreas seems to better cope with the toxins, as no LPO was observed. However, the mechanisms involved in hepatopancreas protection were not enough to maintain DNA integrity. The absence of LPO, and the antioxidant system low responsiveness, suggests DNA damage was not oxidative. When exposed to toxic algae under W, toxin-modulated antioxidant responses were observed in both gills and hepatopancreas. Simultaneous exposure to the stressors highlighted gills susceptibility with a synergistic interaction increasing DNA damage. Exposure to toxic algae under A led to genotoxicity potentiation in both organs. The combined effect of WA did not cause relevant interactions in gills antioxidant responses, but stressors interactions impacted LPO and GDI. Antioxidant responses and LPO pointed out to be modulated by the environmental conditions in hepatopancreas, while GDI results support the dominance of toxin-triggered process. Overall, these results reveal that simultaneous exposure to warming, acidification and PSTs impairs mussel DNA integrity, compromising the genetic information due to the synergetic effects. Finally, this study highlights the increasing ecological risk of harmful algal blooms to Mytilus galloprovinciallis populations.
Subject(s)
DNA Damage , Marine Toxins/toxicity , Mytilus/drug effects , Oxidative Stress/drug effects , Water Pollutants, Chemical/toxicity , Animals , Antioxidants/metabolism , Biomarkers/metabolism , Comet Assay , Dinoflagellida/metabolism , Gills/drug effects , Gills/metabolism , Hepatopancreas/drug effects , Hepatopancreas/metabolism , Hydrogen-Ion Concentration , Lipid Peroxidation/drug effects , Marine Toxins/metabolism , Mytilus/genetics , Mytilus/metabolism , TemperatureABSTRACT
DNA integrity and stability are essential to organisms' health and survival. However, it has been neglected in what concerns to fish farming, disregarding the potential impact of endogenous/ exogenous factors. As marine macroalgae constitute a source of natural compounds with a large spectrum of biological activities, this study, situated in the interface of nutritional-genetic research and development of algae practical applications, aimed to evaluate the genoprotective properties of a macroalgae-enriched diet (total percentage of 5%, incorporating equal percentages of Ulva rigida, Gracilaria gracilis and Fucus vesiculosus) in gilthead seabream (Sparus aurata). Protection was assessed in relation to a basal genome integrity and against an exogenous genotoxic challenge (cyclophosphamide; CP). Fish were reared for 30â¯days with the supplemented diet, being then injected with CP and sampled at days 3 and 10 post-injection (p.i.). To evaluate whether the favorable effects remain after the end of supplementation, a fish subgroup previously fed with algae-enriched diet was submitted to a diet reversion at day 3 p.i., being thereafter fed with the standard diet. Genetic damage was evaluated through the erythrocytic nuclear abnormalities (ENA) and comet assays and complemented by the assessment of the antioxidant system. Results pointed out that algae-enriched feed exhibits anti-genotoxic properties, mostly expressed in relation to the exogenous pressure, manifest in relation to DNA strand breaks and chromosomal lesions, also reducing oxidative DNA damage. Nonetheless, blood antioxidants were only punctually altered by the supplemented diet (e.g. catalase and glutathione-S-transferase). Analyzing the effect persistence, it was perceived that 7â¯days without algae uptake was enough to partially reduce the protection efficacy. Overall, these findings are promising towards the benefits of macroalgae inclusion in fish diet, and thus, to invigorate mariculture activity and the commercial use of algae, also providing new insights on the DNA protection mechanisms.
Subject(s)
Animal Feed , DNA Damage/drug effects , Diet/veterinary , Sea Bream/physiology , Seaweed , Animal Nutritional Physiological Phenomena , AnimalsABSTRACT
This study examined the susceptibility of fish (Liza aurata) eyes and brain to metals(loids) contamination under realistic exposure conditions. A multidimensional approach was applied to fish caught at a chronically contaminated site (BAR) and at a reference site of the Tagus estuary (Portugal), which comprised metals(loids) accumulation in eyes and brain together with a battery of enzymatic and non-enzymatic antioxidants, as well as brain morphometry (i.e. cell density). Trace element levels in the blood, gills, liver and kidney allowed interpretations on their preferential pathway(s) to the eyes and brain. Metals(loids) accumulation pointed out the elevated vulnerability of the fish eyes at BAR, probably related with the direct waterborne uptake. Pb uptake in L. aurata eyes could be associated both with water and indirect pathways. At the most contaminated site, metals(loids) were on the basis of pro-oxidant conditions in the ocular tissues, while no indication of toxicity was recorded in the brain. Overall, the results disclosed a differential bioaccumulation among fish organs, suggesting that, in the L. aurata population studied, metal organotropism underlie the lower susceptibility of the brain comparing to the eyes. However, mechanisms remain little understood and further work is needed.
Subject(s)
Brain/drug effects , Environmental Monitoring , Estuaries , Eye/drug effects , Fishes/physiology , Metals/toxicity , Water Pollutants, Chemical/toxicity , Animals , PortugalABSTRACT
The responses of non-target organisms to pesticide exposure are still poorly explored in what concerns the development of adjustments favouring population success. Owing to the vital role of DNA integrity, it is important to identify genome-maintenance skills and their determinant factors. Thus, the major aims of the present study were: (i) to assess the genotoxicity of the penoxsulam-based herbicide (Viper®) to the crayfish Procambarus clarkii; (ii) to understand the influence of gender and contamination history in the genotoxic responses following exposure to this herbicide; (iii) to investigate the damage mechanisms involved in putative adjustments shown by P. clarkii. Two populations were tested, one from a reference site and the other from a historically contaminated site. Specimens from both populations were exposed to Viper®, considering environmentally relevant penoxsulam concentrations (20 and 40 µg L-1) and to a model genotoxicant (EMS). Comet assay was adopted to assess the genetic damage in gills. The results disclosed the genotoxicity of the herbicide to crayfish (a non-target organism). Additionally, organisms exposed to the highest concentration of penoxsulam signalized the influence of factor "population" towards the genotoxic pressure (measured as effective DNA breaks): P2 males from the historically impacted population displayed a significantly higher susceptibly (by up to 53.98%) when compared to control, while the homologous group from the reference population presented levels similar to its respective control. When DNA lesion-repair enzymes were considered, DNA oxidation patterns suggested an increased ability of this gender (39.75% lower than negative control) to deal with this particular type of damage, namely considering pyrimidines oxidation. It is worth remarking that the influence of the exposure history on the protection/vulnerability to the penoxsulam-based herbicide was only evident in males, despite depending on the type of DNA damage: when the non-specific damage was considered, organisms from the impacted population seemed to be more vulnerable while regarding to the oxidative damage, males from the impacted population appeared to be more protected than organisms that have never been exposed to penoxsulam. Overall, the influence of factors "gender" and "contamination history" was demonstrated as well as its dependence on DNA damage type was evident. EMS groups did not present the differences between populations, reinforcing the agent-specific adjustment hypothesis.These findings highlighted the importance of considering differential physiological backgrounds in ecogenotoxicological analysis, hence favouring the elaboration of more plausible and holistic approaches integrating the environmental risk assessment of pesticides.
Subject(s)
Astacoidea/drug effects , DNA Damage , Herbicides/toxicity , Sulfonamides/toxicity , Uridine/analogs & derivatives , Water Pollutants, Chemical/toxicity , Animals , Comet Assay , Dose-Response Relationship, Drug , Female , Male , Sex Factors , Uridine/toxicityABSTRACT
Enchytraeids have been used in standard ecotoxicity testing for approximately 20 yr. Since adopting the standard test for survival and reproduction, a number of additional tools have been developed, including transcriptomics and enzymatic biomarkers. So far, a genotoxicity tool and endpoint have not been used; hence, the goals of the present study included optimization of the in vivo alkaline comet assay in Enchytraeus crypticus. Further, the effect of silver nanomaterial (Ag NM300K, dispersed, 15 nm) was tested and compared with silver nitrate. Hydrogen peroxide was used as a positive control. The various steps were optimized. The fully detailed standard operating procedure is presented. Silver materials caused genotoxicity, this being differentiated for the nano and non-nano forms. Silver nitrate caused genotoxicity after 3 d of exposure in a dose-related manner, although after 7 d the effects were either reduced or repaired. Ag NM300K caused higher genotoxicity after 7 d for the lowest concentration, highlighting a potential nonmonotonic dose-response effect. Overall, the comet assay showed the power to discriminate effects between materials and also toxicity at low relevant doses. Environ Toxicol Chem 2018;37:184-191. © 2017 SETAC.
Subject(s)
Comet Assay/methods , Mutagens/toxicity , Nanostructures/toxicity , Oligochaeta/drug effects , Silver Nitrate/toxicity , Animals , DNA DamageABSTRACT
The formulation Mancozan®, containing mancozeb as active ingredient, is among the most widely used fungicides. Although mancozeb has been detected in surface waters, studies addressing the genotoxic risk to fish arising from the use of this formulation, testing environmentally realistic concentrations, are absent from the literature. Hence, this work aimed to investigate the DNA and chromosome damaging potential of Mancozan® (0.29 and 2.9µgL-1) in the European eel (Anguilla anguilla L.), after a short-term exposure (3days), through the adoption of the comet and the erythrocytic nuclear abnormality (ENA) assays. In addition, it was intended to elucidate the subjacent damage mechanisms, improving the comet assay with the adoption of the endonucleases formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII), which detect oxidized bases. The highest Mancozan® concentration was able to affect the DNA integrity (comet assay), while the adoption of endonucleases pointed out an oxidative cause to the damage. Regarding the chromosomal damage (ENA assay), both concentrations displayed significant effects, revealing the clastogenic and/or aneugenic properties of Mancozan®. Furthermore, the two genotoxic endpoints were significantly correlated. Overall, the results revealed a genetic hazard to fish inhabiting aquatic systems contaminated by Mancozan® and strongly recommend the development of biomonitoring and regulatory policies regarding the utilization of this agrochemical.
Subject(s)
Anguilla/genetics , DNA Damage , Fungicides, Industrial/toxicity , Maneb/toxicity , Water Pollutants, Chemical/toxicity , Zineb/toxicity , Animals , Chromosomes/drug effects , Comet Assay , DNA-Formamidopyrimidine Glycosylase/metabolism , Endodeoxyribonucleases/metabolism , Erythrocytes/drug effectsABSTRACT
The chronic exposure of benthic organisms to metals in sediments can lead to the development of tolerance mechanisms, thus diminishing their responsiveness. This study aims to evaluate the accumulation profiles of V, Cr, Co, Ni, As, Cd, Pb and Hg and antioxidant system responses of two benthic organisms (Cerastoderma edule, Bivalvia; Nephtys hombergii, Polychaeta). This approach will provide clarifications about the ability of each species to signalise metal contamination. Organisms of both species were collected at the Tagus estuary, in two sites with distinct contamination degrees (ALC, slightly contaminated; BAR, highly contaminated). Accordingly, C. edule accumulated higher concentrations of As, Pb and Hg at BAR compared to ALC. However, antioxidant responses of C. edule were almost unaltered at BAR and no peroxidative damage occurred, suggesting adjustment mechanisms to the presence of metals. In contrast, N. hombergii showed a minor propensity to metal accumulation, only signalising spatial differences for As and Pb and accumulating lower concentrations of metals than C. edule. The differences in metal accumulation observed between species might be due to their distinctive foraging behaviour and/or the ability of N. hombergii to minimise the metal uptake. Despite that, the accumulation of As and Pb was on the basis of the polychaete antioxidant defences inhibition at BAR, including CAT, SOD, GR and GPx. The integrated biomarker response index (IBRv2) confirmed that N. hombergii was more affected by metal exposure than C. edule. In the light of current findings, in field-based studies, the information of C. edule as a bioindicator should be complemented by that provided by another benthic species, since tolerance mechanisms to metals can hinder a correct diagnosis of sediment contamination and of the system's health. Overall, the present study contributed to improve the lack of fundamental knowledge of two widespread and common estuarine species, providing insights of the metal accumulation profiles under a scenario of chronic contamination. Finally, this work provided useful information that can be applied in the interpretation of future environmental monitoring studies.
Subject(s)
Cardiidae/physiology , Environmental Monitoring , Metals/toxicity , Oxidative Stress , Polychaeta/physiology , Water Pollutants, Chemical/toxicity , Animals , Biomarkers/metabolism , Cardiidae/metabolism , Metallothionein/metabolism , Metals/metabolism , Polychaeta/metabolism , Water Pollutants, Chemical/metabolismABSTRACT
Oxidative stress has been described as a key pathway to initiate mercury (Hg) toxicity in fish. However, the mechanisms underlying Hg-induced oxidative stress in fish still need to be clarified. To this aim, environmental metabolomics in combination with a battery of conventional oxidative stress biomarkers were applied to the gills of golden grey mullet (Liza aurata) collected from Largo do Laranjo (LAR), a confined Hg contaminated area, and São Jacinto (SJ), selected as reference site (Aveiro Lagoon, Portugal). Higher accumulation of inorganic Hg and methylmercury was found in gills of fish from LAR relative to SJ. Nuclear magnetic resonance (NMR)-based metabolomics revealed changes in metabolites related to antioxidant protection, namely depletion of reduced glutathione (GSH) and its constituent amino acids, glutamate and glycine. The interference of Hg with the antioxidant protection of gills was corroborated through oxidative stress endpoints, namely the depletion of glutathione peroxidase and superoxide dismutase activities at LAR. The increase of total glutathione content (reduced glutathione+oxidized glutathione) at LAR, in parallel with GSH depletion aforementioned, indicates the occurrence of massive GSH oxidation under Hg stress, and an inability to carry out its regeneration (glutathione reductase activity was unaltered) or de novo synthesis. Nevertheless, the results suggest the occurrence of alternative mechanisms for preventing lipid peroxidative damage, which may be associated with the enhancement of membrane stabilization/repair processes resulting from depletion in the precursors of phosphatidylcholine (phosphocholine and glycerophosphocholine), as highlighted by NMR spectroscopy. However, the observed decrease in taurine may be attributable to alterations in the structure of cell membranes or interference in osmoregulatory processes. Overall, the novel concurrent use of metabolomics and conventional oxidative stress endpoints demonstrated to be sensitive and effective towards a mechanistically based assessment of Hg toxicity in gills of wild fish, providing new insights into the toxicological pathways underlying the oxidative stress.
Subject(s)
Mercury/metabolism , Smegmamorpha/metabolism , Water Pollutants, Chemical/metabolism , Animals , Biological Assay , Biomarkers/metabolism , Gills/drug effects , Gills/metabolism , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Mercury/toxicity , Metabolome/physiology , Metabolomics , Oxidative Stress , Portugal , Proton Magnetic Resonance Spectroscopy , Water Pollutants, Chemical/toxicityABSTRACT
This study aims to contribute to fill a knowledge gap related with Hg effects in fish eyes. As a pioneering strategy, Hg bioaccumulation in eye wall of the wild grey mullet (Liza aurata) was assessed, together with oxidative stress and synaptic transmission profiles. This approach was complemented by the characterisation of environmental contamination (both in water and sediment). Sampling was conducted in winter and summer in two sites of a Portuguese coastal lagoon (Aveiro lagoon): Largo do Laranjo (LAR) - located in an Hg contaminated/confined area; São Jacinto (SJ) - closer to the lagoon inlet and selected as reference site. Levels of total Hg (tHg), inorganic Hg (iHg) and methylmercury (MeHg) in eye wall were higher at LAR than SJ, both in winter and summer, reflecting the environmental contamination patterns. Moreover, fish caught at LAR in winter showed a significant decrease of catalase and superoxide dismutase activities, in line with the occurrence of peroxidative damage. A different spatial pattern was recorded in summer, being characterised by the increment of glutathione peroxidase and glutathione reductase activities at LAR, as well as total glutathione content, preventing the occurrence of lipid peroxidation. Also in summer, a significant decrease of acetylcholinesterase activity was recorded in fish eyes at LAR, pointed out Hg as an anticholinergic agent. Besides Hg, water salinity had probably an indirect effect on spatial and winter-summer variation patterns of AChE. Current data pointed out that Hg (in iHg and MeHg forms) could exert ocular toxicity both by the promotion of oxidative stress and by the interference with neurotransmission processes.
Subject(s)
Eye Diseases/veterinary , Fish Diseases/chemically induced , Mercury/toxicity , Water Pollutants, Chemical/toxicity , Animals , Atlantic Ocean , Eye/chemistry , Eye Diseases/chemically induced , Fishes , Geologic Sediments/chemistry , Seawater/chemistryABSTRACT
Triclopyr-based herbicides are broadly used worldwide for site preparation and forest vegetation management. Thus, following application, these agrochemicals can inadvertently reach the aquatic ecosystems. Garlon(®) is one of the most popular commercial denominations of this group of herbicides, considered as highly toxic to fish, even by its manufacturer. Although DNA is frequently regarded as a target of pesticide toxicity, the genotoxic potential of Garlon(®) to fish remains completely unknown. Hence, the main goal of this study was to evaluate the genotoxicity of Garlon(®) and its active ingredient (triclopyr), clarifying the underlying mechanisms. Therefore, the comet assay, implemented as the standard procedure, with an extra step involving DNA lesion-specific repair enzymes (formamidopyrimidine DNA glycosylase and endonuclease III), was used to identify DNA damage in blood cells of Anguilla anguilla L. Short-term exposures (1 and 3 days) to Garlon(®) and triclopyr were carried out, adopting environmentally realistic concentrations (67.6 and 270.5 µg L(-1) Garlon(®) and 30 and 120 µg L(-1) triclopyr). The results concerning the nonspecific DNA damage proved the risk of the herbicide Garlon(®) and its active ingredient triclopyr in both tested concentrations and exposure lengths. In addition, the higher genotoxic potential of the formulation, in comparison with the active ingredient, was demonstrated. When the additional breaks corresponding to net enzyme-sensitive sites were considered, none of the conditions revealed significant levels of oxidative damage. This identification of the genotoxic properties of triclopyr-based herbicides to fish highlights the need to develop less hazardous formulations, as well as the adoption of mitigation measures related to the application of these agrochemicals in the framework of forestry and agriculture sustainable management.