Showing papers in "Comparative Biochemistry and Physiology C-toxicology & Pharmacology in 2017"

Imazalil exposure induces gut microbiota dysbiosis and hepatic metabolism disorder in zebrafish

Abstract: The fungicide imazalil (IMZ) is used extensively to preserve freshness, prevent decay and control fungal infections in fruits, vegetables or other plants. Recently, some studies have reported that the real in aquatic systems have reached very high levels. Here, male adult zebrafish were exposed to 100 and 1000μg/L IMZ for 1, 7, 21days, and the gut microbiota and hepatic metabolism were evaluated. Exposure to a high concentration of IMZ for 21days decreased mucin secretion in the gut. Sequencing of the V3-V4 region of the bacterial 16S rRNA gene revealed a significant increase in the diversity of gut microbiota in male zebrafish. At the phylum level, the composition of Proteobacteria and Bacteroidetes was decreased, while those Fusobacteria and Firmicutes increased in the gut after exposure to 1000μg/L IMZ for 21days. At the genus level, 29 species of microorganisms were significantly changed after IMZ exposure. Based on GC/MS metabolomics analysis, 101 metabolites were observably significantly altered in the 1000μg/L IMZ-treatment group. These changed metabolites were mainly associated with the pathway of glycolysis, amino acid metabolism, and lipid metabolism. In addition, the transcription of some genes related to glycolysis and lipid metabolism, including Aco, Cpt1, Acc1, Srebp1a and Fas, was decreased significantly in the liver of zebrafish when exposed to 100 and 1000μg/L IMZ for 7 or 21days. These results indicated that exposure to IMZ could cause gut microbiota dysbiosis and metabolic disorders in adult zebrafish.

Are whale sharks exposed to persistent organic pollutants and plastic pollution in the Gulf of California (Mexico)? First ecotoxicological investigation using skin biopsies

Abstract: The whale shark (Rhincodon typus) is an endangered species that may be exposed to micro- and macro-plastic ingestion as a result of their filter-feeding activity, particularly on the sea surface. In this pilot project we perform the first ecotoxicological investigation on whale sharks sampled in the Gulf of California exploring the potential interaction of this species with plastic debris (macro-, micro-plastics and related sorbed contaminants). Due to the difficulty in obtaining stranded specimens of this endangered species, an indirect approach, by skin biopsies was used for the evaluation of the whale shark ecotoxicological status. The levels of organochlorine compounds (PCBs, DDTs), polybrominated diphenyl ethers (PBDEs) plastic additives, and related biomarkers responses (CYP1A) were investigated for the first time in the whale shark. Twelve whale shark skin biopsy samples were collected in January 2014 in La Paz Bay (BCS, Mexico) and a preliminary investigation on microplastic concentration and polymer composition was also carried out in seawater samples from the same area. The average abundance pattern for the target contaminants was PCBs>DDTs>PBDEs>HCB. Mean concentration values of 8.42ng/g w.w. were found for PCBs, 1.31ng/g w.w. for DDTs, 0.29ng/g w.w. for PBDEs and 0.19ng/g w.w. for HCB. CYP1A-like protein was detected, for the first time, in whale shark skin samples. First data on the average density of microplastics in the superficial zooplankton/microplastic samples showed values ranging from 0.00items/m3 to 0.14items/m3. A focused PCA analysis was performed to evaluate a possible correlation among the size of the whale sharks, contaminants and CYP1A reponses. Further ecotoxicological investigation on whale shark skin biopsies will be carried out for a worldwide ecotoxicological risk assessment of this endangerd species.

Gene expression biomarkers of heat stress in scleractinian corals: Promises and limitations

Abstract: Gene expression biomarkers (GEBs) are emerging as powerful diagnostic tools for identifying and characterizing coral stress. Their capacity to detect sublethal stress prior to the onset of signs at the organismal level that might already indicate significant damage makes them more precise and proactive compared to traditional monitoring techniques. A high number of candidate GEBs, including certain heat shock protein genes, metabolic genes, oxidative stress genes, immune response genes, ion transport genes, and structural genes have been investigated, and some genes, including hsp16, Cacna1, MnSOD, SLC26, and Nf-kB, are already showing excellent potential as reliable indicators of thermal stress in corals. In this mini-review, we synthesize the current state of knowledge of scleractinian coral GEBs and highlight gaps in our understanding that identify directions for future work. We also address the underlying sources of variation that have sometimes led to contrasting results between studies, such as differences in experimental set-up and approach, intrinsic variation in the expression profiles of different experimental organisms (such as between different colonies or their algal symbionts), diel cycles, varying thermal history, and different expression thresholds. Despite advances in our understanding there is still no universally accepted biomarker of thermal stress, the molecular response of corals to heat stress is still unclear, and biomarker research in Symbiodinium still lags behind that of the host. These gaps should be addressed in future work.

Correlation between ontogenetic dietary shifts and venom variation in Australian brown snakes (Pseudonaja).

Abstract: Venom is a key evolutionary trait, as evidenced by its widespread convergent evolution across the animal kingdom. In an escalating prey-predator arms race, venoms evolve rapidly to guarantee predatory or defensive success. Variation in venom composition is ubiquitous among snakes. Here, we tested variation in venom activity on substrates relevant to blood coagulation among Pseudonaja (brown snake) species, Australian elapids responsible for the majority of medically important human envenomations in Australia. A functional approach was employed to elucidate interspecific variation in venom activity in all nine currently recognised species of Pseudonaja. Fluorometric enzymatic activity assays were performed to test variation in whole venom procoagulant activity among species. Analyses confirmed the previously documented ontogenetic shift from non-coagulopathic venom in juveniles to coagulopathic venom as adults, except for the case of P. modesta, which retains non-coagulopathic venom as an adult. These shifts in venom activity correlate with documented ontogenetic shifts in diet among brown snakes from specialisation on reptilian prey as juveniles (and throughout the life cycle of P. modesta), to a more generalised diet in adults that includes mammals. The results of this study bring to light findings relevant to both clinical and evolutionary toxinology.

The detoxification responses, damage effects and bioaccumulation in the scallop Chlamys farreri exposed to single and mixtures of benzo[a]pyrene and chrysene.

Abstract: This study aimed to investigate the detoxification responses, damage effects and biotransformation in scallop Chlamys farreri exposed to benzo[a]pyrene (BaP) (0.1, 1μg/L), chrysene (CHR) (0.1, 1μg/L) and BaP+CHR (0.1+0.1, 1+1μg/L) for 15days. Results demonstrated that BaP and CHR concentration (BaP

An AOP analysis of selective serotonin reuptake inhibitors (SSRIs) for fish.

Abstract: Pharmaceuticals and personal care products (PPCPs) are found in measureable quantities within the aquatic environment. Selective serotonin reuptake inhibitor (SSRI) antidepressants are one class of pharmaceutical compound that has received a lot of attention. Consistent with most PPCPs, the pharmacokinetics and physiological impacts of SSRI treatment have been well-studied in small mammals and humans and this, combined with the evolutionary conservation of the serotonergic system across vertebrates, allows for the read-across of known SSRI effects in mammals to potential SSRI impacts on aquatic organisms. Using an Adverse Outcome Pathway (AOP) framework, this review examines the similarities and differences between the mammalian and teleost fish SSRI target, the serotonin transporter (SERT; SLC6A4), and the downstream impacts of elevated extracellular serotonin (5-HT; 5-hydroxytryptamine), the consequence of SERT inhibition, on organ systems and physiological processes within teleost fish. This review also intends to reveal potentially understudied endpoints for SSRI toxicity based on what is known to be controlled by 5-HT in fish.

Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish

Abstract: In recognition of the importance of zebrafish as a model organism for studying human disease, we have created zebrafish content for a web-based reference atlas of microanatomy for comparing histology and histopathology between model systems and with humans (http://bio-atlas.psu.edu). Fixation, decalcification, embedding, and sectioning of zebrafish were optimized to maximize section quality. A comparison of protocols involving six fixatives showed that 10% Neutral Buffered Formalin at 21°C for 24h yielded excellent results. Sectioning of juveniles and adults requires bone decalcification; EDTA at 0.35M produced effective decalcification in 21-day-old juveniles through adults (≥~3Months). To improve section plane consistency in sets of larvae, we have developed new array casting molds based on the outside contours of larvae derived from 3D microCT images. Tissue discontinuity in sections, a common barrier to creating quality sections of zebrafish, was minimized by processing and embedding the formalin-fixed zebrafish tissues in plasticized forms of paraffin wax, and by periodic hydration of the block surface in ice water between sets of sections. Optimal H&E (Hematoxylin and Eosin) staining was achieved through refinement of standard protocols. High quality slide scans produced from glass histology slides were digitally processed to maximize image quality, and experimental replicates posted as full slides as part of this publication. Modifications to tissue processing are still needed to eliminate the need for block surface hydration. The further addition of slide collections from other model systems and 3D tools for visualizing tissue architecture would greatly increase the utility of the digital atlas.

Cardio-respiratory function during exercise in the cobia, Rachycentron canadum: The impact of crude oil exposure.

Abstract: Aerobic exercise capacity is dependent on the cardiorespiratory system's ability to supply oxygen at a rate that meets energetic demands. In teleost fish crude oil exposure, with the associated polycyclic aromatic hydrocarbons (PAH's), reduces exercise performance and this has been hypothesized to be due to compromised cardiovascular function. In this study, we test this hypothesis by simultaneously measuring cardiovascular performance, oxygen consumption, and swim performance in a pelagic teleost, the cobia (Rachycentron canadum). Metabolic rate increased over 300% in both groups during the swim trial but as the fish approached the critical swim speed (Ucrit) MO2 was 12% lower in the oil exposed fish. Further, stroke volume was initially 35% lower while heart rate was 15% higher in the oil exposed compared to control fish. Our findings suggested, while aspects of cardiovascular and metabolic function are altered by oil exposure, additional studies are needed to further understand the homeostatic mechanisms that may sustain cardiovascular function at higher exercise intensities in cobia.

Review of the photo-induced toxicity of environmental contaminants.

Abstract: Solar radiation is a vital component of ecosystem function. However, sunlight can also interact with certain xenobiotic compounds in a phenomenon known as photo-induced, photo-enhanced, photo-activated, or photo-toxicity. This phenomenon broadly refers to an interaction between a chemical and sunlight resulting in increased toxicity. Because most aquatic ecosystems receive some amount of sunlight, co-exposure to xenobiotic chemicals and solar radiation is likely to occur in the environment, and photo-induced toxicity may be an important factor impacting aquatic ecosystems. However, photo-induced toxicity is not likely to be relevant in all aquatic systems or exposure scenarios due to variation in important ecological factors as well as physiological adaptations of the species that reside there. Here, we provide an updated review of the state of the science of photo-induced toxicity in aquatic ecosystems.

Assessment of 8-hydroxy-2-deoxyguanosine activity, gene expression and antioxidant enzyme activity on rainbow trout (Oncorhynchus mykiss) tissues exposed to biopesticide.

Abstract: The goal of this study was to determinate toxicity mechanism of biopesticide with antioxidant enzymes parameters such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and malondialdehyde (MDA) levels, oxidative DNA damage (8-hydroxy-2-deoxyguanosine (8-OHdG)), transcriptional changes of heat shock protein 70 (HSP70), and cytochromes P4501A (CYP1A), sod, cat, and gpx in liver and gill tissues of Oncorhynchus mykiss. For this aim, plant-based (natural pesticides, azadirachtin (AZA)) and synthetic pesticides (deltamethrin (DLM)) were exposed on the fish at different concentrations (0.0005 and 0.00025ppm of DLM; 0.24 and 0.12ppm of AZA) for 21 days. According to the results of the study, the activity of SOD, CAT and GPx decreased, but malondialdehyde (MDA) level and activity of 8-OHdG increased in the gill and liver of rainbow trout (p<0.05). Additionally sod, cat and gpx were down regulated; HSP70 and CYP1A were up regulated for transcriptional observation. The downwards regulation of antioxidant (sod, cat and gpx) and the upregulation of HSP70 and CYP1A was obvious with doses of AZA or DLM (p<0.05). The findings of this study suggest that biopesticide can cause biochemical and physiological effects in the fish gill and liver by causing enzyme inhibition, an increase in 8-OHdG levels and changes in both transcriptional parameters (sod, cat, gpx, HSP70 and CYP1A). We found that excessive doses of plant-based pesticide are nearly as toxic as chemical ones for aquatic organisms. Moreover, 8-OHdG, HSP70 and CYP1A used as a biomarker to determinate toxicity mechanism of biopesticide in aquatic environment.

Ameliorative effect of propolis supplementation on alleviating bisphenol-A toxicity: Growth performance, biochemical variables, and oxidative stress biomarkers of Nile tilapia, Oreochromis niloticus (L.) fingerlings.

Abstract: Bisphenol-A (BPA) is one of the important pollutants in aquatic ecosystems and its detrimental effect on fish has a great concern. Propolis is a natural immune-stimulant that has various biological and pharmacological activities. Thus, its capability to alleviate the toxic effect of BPA on Nile tilapia, Oreochromis niloticus (L.) performance was assessed in a study based on a 2×2 factorial design with two levels of ethanolic extract of propolis (EEP) and two waterborne BPA concentrations in triplicates. Fish (33.9±0.55g) were exposed to 0.0 or 1.64μgBPA/L for 6weeks during which fish were fed on diets containing 0.0 or 9.0gEEP/kg diet. Fish performance, biochemical variables, and oxidative stress enzymes were significantly affected by propolis supplementation, BPA exposure, and their interaction. Propolis supplementation significantly improved fish growth and feed intake, which were significantly retarded by BPA exposure. Additionally, total protein, albumin, globulin, and acetylcholine esterase (AChE) decreased significantly. Meanwhile aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), creatinine, and uric acid increased significantly with exposure to BPA. Levels of malondialdehyde (MDA) as well as superoxide dismutase (SOD) and catalase (CAT) activities increased significantly due to BPA exposure, whereas significant reductions in the activity of glutathione peroxidase (GPx) and glutathione S-transferase (GST) were also recorded compared to the control fish. It is noticed that EEP co-administration ameliorated these parameters. The present results evoked that propolis administration improves fish growth and alleviated BPA-induced toxicity.

A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae.

Abstract: Mitochondria, an integral component of cellular energy metabolism and other key functions, are extremely vulnerable to damage by environmental stressors. Although methods to measure mitochondrial function in vitro exist, sensitive, medium- to high-throughput assays that assess respiration within physiologically-relevant whole organisms are needed to identify drugs and/or chemicals that disrupt mitochondrial function, particularly at sensitive early developmental stages. Consequently, we have developed and optimized an assay to measure mitochondrial bioenergetics in zebrafish larvae using the XF e 24 Extracellular Flux Analyzer. To prevent larval movement from confounding oxygen consumption measurements, we relied on MS-222-based anesthetization. We obtained stable measurement values in the absence of effects on average oxygen consumption rate and subsequently optimized the use of pharmacological agents for metabolic partitioning. To confirm assay reproducibility we demonstrated that triclosan, a positive control, significantly decreased spare respiratory capacity. We then exposed zebrafish from 5 hours post-fertilization (hpf) to 6 days post-fertilization (dpf) to three polycyclic aromatic hydrocarbons (PAHs) – benzo( a )pyrene (BaP), phenanthrene (Phe), and fluoranthene (FL) – and measured various fundamental parameters of mitochondrial respiratory chain function, including maximal respiration, spare respiratory capacity, mitochondrial and non-mitochondrial respiration. Exposure to all three PAHs decreased spare respiratory capacity and maximal respiration. Additionally, Phe exposure increased non-mitochondrial respiration and FL exposure decreased mitochondrial respiration and increased non-mitochondrial respiration. Overall, this whole organism-based assay provides a platform for examining mitochondrial dysfunction in vivo at critical developmental stages. It has important implications in biomedical sciences, toxicology and ecophysiology, particularly to examine the effects of environmental chemicals and/or drugs on mitochondrial bioenergetics.

Catch a tiger snake by its tail: Differential toxicity, co-factor dependence and antivenom efficacy in a procoagulant clade of Australian venomous snakes.

Abstract: A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This study examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels of cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

Abstract: While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

Acute toxicity, bioaccumulation and effects of dietary transfer of silver from brine shrimp exposed to PVP/PEI-coated silver nanoparticles to zebrafish.

Abstract: The extensive use and release to the aquatic environment of silver nanoparticles (NPs) could lead to their incorporation into the food web. Brine shrimp larvae of 24h showed low sensitivity to the exposure to PVP/PEI-coated Ag NPs (5nm), with EC50 values at 24h of 19.63mgAgL-1, but they significantly accumulated silver after 24h of exposure to 100μgL-1 of Ag NPs. Thus, to assess bioaccumulation and effects of silver transferred by the diet in zebrafish, brine shrimp larvae were exposed to 100ngL-1 of Ag NPs as an environmentally relevant concentration or to 100μgL-1 as a potentially effective concentration and used to feed zebrafish for 21days. Autometallography revealed a dose- and time-dependent metal accumulation in the intestine and in the liver of zebrafish. Three-day feeding with brine shrimps exposed to 100ngL-1 of Ag NPs was enough to impair fish health as reflected by the significant reduction of lysosomal membrane stability and the presence of vacuolization and necrosis in the liver. However, dietary exposure to 100μgL-1 of Ag NPs for 3days did not significantly alter gene transcription levels, neither in the liver nor in the intestine. After 21days, biological processes such as lipid transport and localization, cellular response to chemical stimulus and response to xenobiotic stimulus were significantly altered in the liver. Overall, these results indicate an effective dietary transfer of silver and point out to liver as the main target organ for Ag NP toxicity in zebrafish after dietary exposure.

The zebrafish operculum: A powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance.

Abstract: Bone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3days initiated at 3days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose-dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.

Suitability of cholinesterase of polychaete Diopatra neapolitana as biomarker of exposure to pesticides: In vitro characterization

Abstract: Cholinesterases of Diopatra neapolitana were characterized for their activity in whole body and different body segments (apical, intermediate, posterior), substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, isoOMPA, BW284C51) and carbamates (carbofuran, methomyl, aldicarb and carbaryl). Results showed that the rate of hydrolysis for acetyl- and propionylthiocholine was higher in the posterior segment than the apical/intermediate segments and whole body. Cholinesterases of D. neapolitana showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was poorly hydrolyzed indicating, together with the absence of inhibition by the specific inhibitor and the absence of reactive bands in native electrophoresis, a lack of an active butyrylcholinesterase, differently than that observed in other Annelida species. The degree of inhibition by selected carbamates of cholinesterase activity with propionylthiocholine as substrate was higher than that observed with ATChI-ChE activity; aldicarb showed the highest inhibitory effect.

Developmental and reproductive toxicity of PVP/PEI-coated silver nanoparticles to zebrafish.

Abstract: Cellular and molecular mechanisms of toxicity of silver nanoparticles (NPs) and their toxicity to fish embryos after waterborne exposure have been widely investigated, but much less information is available regarding the effect of Ag NPs on physiological functions such as growth or reproduction. In this work, the effects of waterborne exposure of adult zebrafish (Danio rerio) to PVP/PEI coated Ag NPs (~5nm) on reproduction (fecundity) were investigated. Moreover, the development of the embryos after parental exposure was compared with the development of embryos after direct waterborne exposure to the NPs. For this, two experiments were run: 1) embryos from unexposed parents were treated for 5days with Ag NPs (10μgAgL-1-10mgAgL-1) and development was monitored, and 2) selected breeding zebrafish were exposed for 3weeks to 100ngAgL-1 (environmentally relevant concentration) or to 10μgAgL-1 of Ag NPs, fecundity was scored and development of resulting embryos was monitored up to 5days. Waterborne exposure of embryos to Ag NPs resulted in being highly toxic (LC50 at 120h=50μgAgL-1), causing 100% mortality during the first 24h of exposure at 0.1mgAgL-1. Exposure of adults, even at the environmentally relevant silver concentration, caused a significant reduction of fecundity by the second week of treatment and resulting embryos showed a higher prevalence of malformations than control embryos. Exposed adult females presented higher prevalence of vacuolization in the liver. These results show that Ag NPs at an environmentally relevant concentration are able to affect population level parameters in zebrafish.

Alteration of neurotransmission and skeletogenesis in sea urchin Arbacia lixula embryos exposed to copper oxide nanoparticles.

Abstract: The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula , an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20 ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N -acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130 , did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota.

Physiological and biochemical impacts of graphene oxide in polychaetes: The case of Diopatra neapolitana.

Abstract: Graphene oxide (GO) is an important carbon nanomaterial (NM) that has been used, but limited literature is available regarding the impacts induced in aquatic organisms by this pollutant and, in particular in invertebrate species. The polychaete Diopatra neapolitana has frequently been used to evaluate the effects of environmental disturbances in estuarine systems due to its ecological and socio-economic importance but to our knowledge no information is available on D. neapolitana physiological and biochemical alterations due to GO exposure. Thus, the present study aimed to assess the toxic effects of different concentrations of GO (0.01; 0.10 and 1.00 mg/L) in D. neapolitana physiological (regenerative capacity) and biochemical (energy reserves, metabolic activity and oxidative stress related biomarkers) performance, after 28 days of exposure. The results obtained revealed that the exposure to GO induced negative effects on the regenerative capacity of D. neapolitana , with organisms exposed to higher concentrations regenerating less segments and taking longer periods to completely regenerate. GO also seemed to alter energy-related responses, especially glycogen content, with higher values in polychaetes exposed to GO which may result from a decreased metabolism (measured by electron transport system activity), when exposed to GO. Furthermore, under GO contamination D. neapolitana presented cellular damage, despite higher activities of antioxidant and biotransformation enzymes in individuals exposed to GO.

A colour preference technique to evaluate acrylamide-induced toxicity in zebrafish.

Abstract: The zebrafish has become a commonly used vertebrate model for toxicity assessment, of particular relevance to the study of toxic effects on the visual system because of the structural similarities shared by zebrafish and human retinae. In this article we present a colour preference-based technique that, by assessing the functionality of photoreceptors, can be used to evaluate the effects of toxicity on behaviour. A digital camera was used to record the locomotor behaviour of individual zebrafish swimming in a water tank consisting of two compartments separated by an opaque perforated wall through which the fish could pass. The colour of the lighting in each compartment could be altered independently (producing distinct but connected environments of white, red or blue) to allow association of the zebrafish's swimming behaviour with its colour preference. The functionality of the photoreceptors was evaluated based on the ability of the zebrafish to sense the different colours and to swim between the compartments. The zebrafish tracking was carried out using our algorithm developed with MATLAB. We found that zebrafish preferred blue illumination to white, and white illumination to red. Acute treatment with acrylamide (2 mM for 36 h) resulted in a marked reduction in locomotion and a concomitant loss of colour-preferential swimming behaviour. Histopathological examination of acrylamide-treated zebrafish eyes showed that acrylamide exposure had caused retinal damage. The colour preference tracking technique has applications in the assessment of neurodegenerative disorders, as a method for preclinical appraisal of drug efficacy and for behavioural evaluation of toxicity.

The expression of P450 genes mediating fenpropathrin resistance is regulated by CncC and Maf in Tetranychus cinnabarinus (Boisduval).

Abstract: Although overexpression of genes encoding detoxification enzymes is a well-known mechanism of pesticide resistance of mites, the regulators involved in this process are still illiterate. Previous studies in our laboratory demonstrated that the overexpression of six P450 genes contributes to fenpropathrin resistance in T. cinnabarinus . In this study, six transcription factor genes that likely regulate the expression of P450 genes were identified and characterized. Quantitative PCR (qPCR) analysis showed that three transcription factor genes were highly expressed in a fenpropathrin-resistant (FeR) strain of T. cinnabarinus . The cap ‘n’ collar isoform C (CncC) and muscle aponeurosis fibromatosis (Maf) family transcription factors were identified as the key regulator of P450 genes by RNA interference (RNAi). Furthermore, research on the promoters of these P450 genes using reporter assays identified that CncC and Maf influence the susceptibility of T. cinnabarinus to fenpropathrin through regulating the expression of P450 genes. This study increases our understanding of the molecular mechanisms underlying the regulation of P450 genes involved in detoxification of acaricides in T. cinnabarinus .

Does pre-exposure to warming conditions increase Mytilus galloprovincialis tolerance to Hg contamination?

Abstract: The degree to which marine invertebrate populations can tolerate extreme weather events, such as short-term exposure to high temperatures, and the underlying biochemical response mechanisms are not yet fully understood Furthermore, scarce information is available on how marine organisms respond to the presence of pollutants after exposure to heat stress conditions Therefore, the present study aimed to understand how the mussel Mytilus galloprovincialis responds to Hg pollution after pre-exposure to warming conditions Mussels were exposed to control (17 °C) and warming (21 °C) conditions during 14 days, followed by Hg contamination during 28 days under different temperature regimes (17 and 21 °C) The results obtained demonstrated significantly higher Hg concentrations in mussels under 17 °C during the entire experiment than in organisms exposed to 21 °C during the same period, which resulted in higher oxidative stress in mussels under control temperature Significantly higher Hg concentrations were also observed in mussels pre-exposed to 21 °C followed by a 17 °C exposure comparing with organisms maintained the entire experiment at 21 °C These results may be explained by higher metabolic capacity in organisms exposed to 17 °C after pre-exposure to 21 °C that although induced antioxidant defences were not enough to prevent oxidative stress No significant differences in terms of Hg concentration were found between mussels exposed to 17 °C during the entire experiment and organisms pre-exposed to 21 °C followed by a 17 °C exposure, leading to similar oxidative stress levels in mussels exposed to both conditions Therefore, our findings demonstrated that pre-exposure to warming conditions did not change mussels' accumulation and tolerance to Hg in comparison to Hg contaminated mussels maintained at control temperature Furthermore, the present study indicate that organisms maintained under warming conditions for long periods may prevent the accumulation of pollutants by decreasing their metabolism which will limit cellular injuries Capsule Mussels under warming conditions presented reduced metabolic capacity, resulting in lower Hg accumulation, which in turn prevented higher damages and, consequently, physiological impairments

Neurotoxic effects, molecular responses and oxidative stress biomarkers in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) exposed to verapamil.

Abstract: Pharmaceutical drugs and their metabolites are detected in aquatic ecosystems and have been reported to cause ecotoxicological consequences to resident aquatic organisms. The study investigated the effects of acute and long-term exposure to verapamil on activities of acetylcholinesterase and antioxidant enzymes as well as mRNA expression of stress-related genes in brain and muscle tissues of Nile tilapia, Oreochromis niloticus. The 96 h LC50 of verapamil to O. niloticus was 2.29 mg L− 1. Exposure to sub-lethal concentrations of verapamil (0.14, 0.29 and 0.57 mg L− 1) for period of 15, 30, 45 and 60 days, led to inhibition of acetylcholinesterase activities in the brain and muscle of the fish. The activities of the oxidative enzymes such as the catalase, superoxide dismutase and glutathione peroxidase were also inhibited in both the tissues while there was an increase in the activities of glutathione–S-transferase and reduced glutathione in the muscle after 15 days at 0.29 mg L− 1. Lipid peroxidation and carbonyl protein showed elevated level, indicating a positive correlation with both time and concentration. The activities of energy-related biomarker (Na+-K+-ATPase) in both the tissues were significantly inhibited (p

Influence of temperature on the antioxidant responses and lipid peroxidation of two species of tadpoles (Rhinella schneideri and Physalaemus nattereri) exposed to the herbicide sulfentrazone (Boral 500SC

Abstract: Amphibians can experience large temperature fluctuations in their habitats, especially during the larval stage, when tadpoles are restricted to small and ephemeral ponds. Changes in water temperature can alter development, metabolism and behaviour of cold-blooded animals but also the toxicokinetics of chemicals in the environment. In Brazil, pesticides application is intensified during the rainy season, which is the period of reproduction for many amphibian species. We evaluated here the influence of temperature (28, 32, and 36 °C) on the toxicity of the herbicide sulfentrazone (Boral®SC) in tadpoles of Physalaemus nattereri and Rhinella schneideri, by analysis of oxidative stress biomarkers. Exposure of tadpoles to sulfentrazone altered the antioxidant enzymes activities and induced lipid peroxidation with temperature-associated responses. Catalase, superoxide dismutase and glucose-6-phosphate dehydrogenase (G6PDH) were impaired by combined effect of temperature and sulfentrazone in both species. G6PDH was increased in most groups exposed to 36 °C. Biotransformation enzyme glutathione-S-transferase had more evident alterations in P. nattereri at higher temperatures and changes in tGSH contents presented different patterns between the species. Lipid peroxidation was particularly induced in tadpoles of P. nattereri. Integrated biomarker response (IBR) index indicated a synergic effect of temperature and sulfentrazone for tadpoles of P. nattereri, while the IBR was mainly influenced by temperature in R. schneideri. Our study showed that temperature modulates biochemical responses in tadpoles exposed to sulfentrazone with a species-specific pattern. These findings imply that the effects of abiotic factors should be taken into account to evaluate the real risks of exposure of amphibians to commonly used pesticides.

Homarus gammarus (Crustacea: Decapoda) larvae under an ocean acidification scenario: responses across different levels of biological organization.

Abstract: The present study evaluated the effects of exposure to different target pCO2 levels: control (C: 370 μatm, pH = 8.15) and ocean acidification (OA: 710 μatm, pH = 7.85) on development and biochemical responses related with oxidative stress and energy metabolism during the crustacean Homarus gammarus (L.) larval development, integrating different levels of biological organization. After hatching in the laboratory, larvae from the same female brood were exposed to the described conditions from hatching until reaching Stage III (last larval stage - 11 days). H. gammarus larvae demonstrated some susceptibility when addressing the predicted pCO2 levels for 2100. Further analysis at the biochemical and physiological level highlighted the occurrence of oxidative stress in the OA scenario (Superoxide Dismutase reduction and higher DNA damage) that was followed by developmental effects, increased inter-moult period from SII to SIII and reduced growth. The extended exposure to these conditions may affect organisms' key life-cycle functions such as physiological resistance, growth, sexual maturation, or reproduction with implications in their future fitness and population dynamics.

Oxidative stress of imidaclothiz on earthworm Eisenia fetida

Abstract: Imidaclothiz, a kind of neonicotinoid insecticides, is developed as a commercial pesticide in China and used for the control of sucking and biting insects, including aphids, whiteflies, beetles and some Lepidoptera species as well. In recent years, more and more attention has been focused on the adverse effects of neonicotinoid insecticides on non-target organisms. In this study, inhibition effects of imidaclothiz and recovery capacity of molecular biomarkers (GST, CE, CAT, SOD and POD activity, and DNA damage) in earthworms were detected for the first time. Earthworms were exposed to imidaclothiz (0.3 and 1.0mgkg-1) for 28days, and then transferred to the clean artificial soil for 28days. Regarding enzyme activities, GST, CE, CAT and POD were inhibited following exposure to imidaclothiz at one or more sample times, while SOD was not affected. After transferring to the clean artificial soil, POD continued to be significantly inhibited till sample time 35days at 0.3mg/kg-1. For GST, CE, CAT and SOD, activities of them were significantly increased compared to those of the control during the recovery period. The olive tail moments (OTM) in comet assay, as an indication of DNA damage, were significantly increased compared to the control after treatment and then decreased during the recovery period. These results suggested that imidaclothiz induced oxidative damage on earthworms which caused damage to vital macromolecules including DNA. However, the recovery changes of all tested biomarkers following the transfer to clean soil suggested the tested earthworm recovery from imidaclothiz exposure.

Utilization of Mytilus digestive gland cells for the in vitro screening of potential metabolic disruptors in aquatic invertebrates.

Abstract: In vertebrate systems, many endocrine disruptors (EDs) can also interfere with energy and lipid metabolism, thus acting as metabolic disruptors. At the cellular level, these effects are mainly mediated by interactions with nuclear receptors/transcription factors, leading to the modulation of genes involved in lipid homeostasis, as well as by rapid, receptor-independent pathways. Several potential metabolic disruptors are found in aquatic environments. In fish, different EDs have been shown to affect hepatic lipid homeostasis both in vivo and in vitro. However, little information is available in aquatic invertebrates due to our poor knowledge of the regulatory pathways of lipid metabolism. In this work, primary cell cultures from the digestive gland of the bivalve Mytilus galloprovincialis were utilized to investigate the effects of model EDs (bisphenol A (BPA) and perfluorooctane sulphonate (PFOS)) on lipid homeostasis. Both compounds (at 24 and 3h of exposure) increased intracellular lipid and tryglyceride-TAG content, with strongest effects of PFOS at 10-7M. Acyl-CoA oxidase activity was unaffected, whereas some changes in the activity of glycolytic, antioxidant/biotransformation enzymes were observed; however, no clear relationship was found with lipid accumulation. Evaluation of mitochondrial membrane potential Δψm and determination of extracellular TAG content indicate that PFOS interferes with mitochondrial function and lipid secretion, whereas BPA mainly affects lipid secretion. Experiments with specific inhibitors showed that activation of PI-3 kinase and extracellularly regulated mitogen-activated protein kinase (ERK MAPK) plays a key role in mediating lipid accumulation. Mussel digestive gland cells represent a simple in vitro model for screening the metabolic effects of EDs in marine invertebrates.

Growth, blood health, antioxidant status, immune response and resistance to Aeromonas hydrophila of juvenile yellow catfish exposed to di-2-ethylhexyl phthalate (DEHP).

Abstract: Triplicate groups of juvenile yellow catfish Pelteobagrus fulvidraco were exposed to three levels of DEHP (0, 0.1 and 0.5mgL-1) for 56days. Fish survival (100%) was not affected by different levels of ambient DEHP. Final body weight, weight gain, specific growth rate and feed intake of fish exposed to 0.5mgL-1 DEHP were the highest. On the contrary, hepatosomatic index of fish exposed to 0.1 and 0.5mgL-1 DEHP were the lowest. Serum total protein, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, glucose and triglycerides increased with the increasing concentrations of DEHP exposure. Superoxide dismutase and glutathione peroxidase activities of fish exposed to 0.5mgL-1 DEHP were the lowest, but malondialdehyde contents of fish exposed to 0.1 and 0.5mgL-1 DEHP were higher than that of control fish. Phagocytic indices of the control group were the highest. After being intraperitoneally injected with Aeromonas hydrophila, fish in the control group had the highest expression of toll like receptor 5, and the expression of myeloid differentiation factor 88 of fish exposed to 0.5mgL-1 DEHP was the lowest. This study indicates that DEHP exerts its toxic effects by interfering with hepatic metabolism, inducing ROS generation and malondialdehyde accumulation, leading to blood deterioration and immunosuppression.

Acute exposure to the penconazole-containing fungicide Topas partially augments antioxidant potential in goldfish tissues.

Abstract: Penconazole is a systemic fungicide commonly used in agriculture as the commercial preparation Topas. Although triazole fungicides are widely found in the aquatic environment, little is known about their acute toxicity on fish. In this study we assessed the effects of short-term exposure to Topas on some parameters of homeostasis of reactive oxygen species (ROS), such as the levels of markers of oxidative stress and parameters of the antioxidant defense system of goldfish (Carassius auratus L.). Gills appeared to be the main target organ of Topas toxicity, showing the greatest number of parameters affected. Gills of Topas-treated fish showed a higher content of low (L-SH) and high (H-SH) molecular mass thiols and higher activities of superoxide dismutase (SOD), catalase, glutathione reductase (GR), glutathione-S-transferase (GST), and glucose-6-phosphate dehydrogenase (G6PDH) as well as reduced carbonyl protein content (CP), as compared with those in the control group. In the liver, goldfish exposure to 15-25mgL-1 Topas resulted in a higher L-SH and H-SH content, but lower CP levels and activity of GST. In kidney, Topas exposure resulted in higher activities of glutathione peroxidase (GPx) and G6PDH, but lower L-SH content and activity of GST. The results of this study indicate that acute goldfish exposure to the triazole fungicide Topas increased efficiency of the antioxidant system in fish gills, liver, and kidney. This could indicate the development of low intensity oxidative stress which up-regulates defense mechanisms responsible for protection of goldfish against deleterious ROS effects.