A novel transcription factor Rwdd1 and its SUMOylation inhibit the expression of sqr, a key gene of mitochondrial sulfide metabolism in Urechis unicinctus Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-20 Xueyu Li, Xiaolong Liu, Zhenkui Qin, Maokai Wei, Xitan Hou, Tingting Zhang, Zhifeng Zhang
Sulfide-quinone oxidoreductase (SQR) is a key enzyme of sulfide metabolism in metazoans, and responsible for oxidizing sulfide into thiosulfate and transmitting the generated electrons to the ubiquinone. It has been revealed that the sqr mRNA level increases significantly in echiuran worm Urechis unicinctus exposed to sulfide, and HSF1, NF1 and Sp1 have been verified to participate in its transcriptional regulation. In this study, we obtained 23 potential transcription factors interacting possibly with the proximal region (-391~+50) of sqr promoter, and focused on the RWD domain-containing 1 (Rwdd1), a protein with the maximum number of clones in yeast one-hybrid (Y1H) screening, to investigate its transcriptional regulation to U. unincitus sqr. The ChIP and EMSA assays identified that the Rwdd1 can bind directly to the promoter (+18/+36) of U. unicinctus sqr. The point mutation and transient transfection experiments discovered that TACG was the key sequence of the DNA element bound by the Rwdd1. Furthermore, the U. unicinctus Rwdd1 (UuRwdd1) was identified to be a transcription repressor inhibiting the sqr promoter activity, and the SUMOylation of UuRwdd1 at the lysine of 90th enhanced its inhibitory effect on sqr transcription further. Western blotting found Rwdd1 responded to sulfide in hindguts from U. unincitus, and the protein content showed a remarkable drop in hindgut nuclei in the early sulfide exposure, and then increased significantly both in the total protein and the nuclear protein extract. We suggested that the Rwdd1 is a novel transcription factor, and these data improve our understanding of the sqr transcriptional regulation and the mitochondrial sulfide metabolism.
Effects of ocean warming and acidification on accumulation and cellular responsiveness to cadmium in mussels Mytilus galloprovincialis: importance of the seasonal status Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-18 Alessandro Nardi, Maura Benedetti, Giuseppe d’Errico, Daniele Fattorini, Francesco Regoli
Ocean warming and acidification could represent an additional threat to marine organisms already coping with other anthropogenic impacts, such as chemical contamination in coastal areas. In this study, interactions between such multiple stressors and their synergistic effects in terms of accumulation, detoxification and biological effects of metals were investigated in the Mediterranean mussel Mytilus galloprovincialis. Organisms sampled during the winter period were exposed for 28 days to different combinations of two temperatures (10 °C and 15 °C), two pH/pCO2 (8.20/~400µatm and 7.4/~3000µatm) and two cadmium concentrations (0 and 20 µg/L). Cadmium concentrations increased in digestive glands and gills of metal-exposed mussels and were further enhanced by co-exposure at higher temperature. Interactive effects of temperature and/or pH were observed on Cd-mediated metallothionein induction, responsiveness of antioxidant system and onset of oxidative damages in lipids, with tissue-specific effects. Immunological effects showed a generalized sensitivity of lysosomal membrane stability toward the investigated stressors with major effects in co-exposed organisms. Cadmium and temperature affected phagocytosis efficiency and composition of haemocyte populations probably influencing the micronucleus frequency through varied mitotic rate. Several differences were highlighted between these results and those previously obtained from mussels exposed in summer, supporting the importance of season when addressing the tolerance of temperate organisms to variations of environmental factors. The elaboration of the whole biomarker results through weighted criteria allowed to summarize specific hazard indices, highlighting tissue-specific sensitivity toward multiple stressors and the need of improving the knowledge on interactions between multiple stressors.
A Temporal High-Resolution Investigation of the Ah-Receptor Pathway during Early Development of Zebrafish (Danio rerio) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-15 Henriette Meyer-Alert, Kim Ladermann, Maria Larsson, Sabrina Schiwy, Henner Hollert, Steffen H. Keiter
In order to contribute to a comprehensive understanding of the regulating mechanisms of the aryl-hydrocarbon-receptor (AHR) in zebrafish embryos, we aimed to elucidate the interaction of proteins taking part in this signaling pathway during early development of the zebrafish (Danio rerio) after chemical exposure. We managed to illustrate initial transcription processes of the implemented proteins after exposure to two environmentally relevant chemicals: polychlorinated biphenyl 126 (PCB126) and β-Naphthoflavone (BNF). Using qPCR, we quantified mRNA every 4 hours until 118 hours post fertilization and found the expression of biotransformation enzymes (cyp1 family) and the repressor of the AHR (AHR-R) to be dependent on the duration of chemical exposure and the biodegradability of the compounds. PCB126 induced persistent increased the amount of transcripts as it is not metabolized, whereas activation by BNF was limited to the initial period of exposure. We did not find a clear relation between the amount of transcripts and activity of the induced cyp-proteins, so posttranscriptional mechanisms are likely to regulate biotransformation of BNF. With regard to zebrafish embryos and their application in risk assessment of hazardous chemicals, our examination of the AHR pathway especially supports the relevance of the time point or period of exposure that is used for bioanalytical investigations and consideration of chemical properties determining biodegradability.
Development of the sea urchin Heliocidaris crasssispina from Hong Kong is robust to ocean acidification and copper contamination Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-12 Narimane Dorey, Elizaldy Maboloc, Kit Yu Karen Chan
Metallic pollution is of particular concern in coastal cities. In the Asian megacity of Hong Kong, despite water qualities have improved over the past decade, some local zones are still particularly affected and could represent sinks for remobilization of labile toxic species such as copper. Ocean acidification is expected to increase the fraction of the most toxic form of copper (Cu2+) by 2.3-folds by 2100 (pH ≈7.7), increasing its bioavailability to marine organisms. Multiple stressors are likely to exert concomitant effects (additive, synergic or antagonist) on marine organisms.Here, we tested the hypothesis that copper contaminated waters are more toxic to sea urchin larvae under future pH conditions. We exposed sea urchin embryos and larvae to two low-pH and two copper treatments (0.1 and 1.0 μM) in three separate experiments. Over the short time typically used for toxicity tests (up to 4-arm plutei, i.e. 3 days), larvae of the sea urchin Heliocidaris crassispina were robust and survived the copper levels present in Hong Kong waters today (≤0.19 μM) as well as the average pH projected for 2100. We, however, observed significant mortality with lowering pH in the longer, single-stressor experiment (Expt A: 8-arm plutei, i.e. 9 days). Abnormality and arm asymmetry were significantly increased by pH or/and by copper presence (depending on the experiment and copper level). Body size (d3; but not body growth rates in Expt A) was significantly reduced by both lowered pH and added copper. Larval respiration (Expt A) was doubled by a decrease at pHT from 8.0 to 7.3 on d6. In Expt B1.0 and B0.1, larval morphology (relative arm lengths and stomach volume) were affected by at least one of the two investigated factors.Although the larvae appeared robust, these sub-lethal effects may have indirect consequences on feeding, swimming and ultimately survival. The complex relationship between pH and metal speciation/uptake is not well-characterized and further investigations are urgently needed to detangle the mechanisms involved and to identify possible caveats in routinely used toxicity tests.
Effects of imidacloprid and a neonicotinoid mixture on aquatic invertebrate communities under Mediterranean conditions Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-12 Andreu Rico, Alba Arenas-Sánchez, Julia Pasqualini, Ariadna García-Astillero, Laura Cherta, Leonor Nozal, Marco Vighi
Neonicotinoid insecticides are considered contaminants of concern due to their high toxicity potential to non-target terrestrial and aquatic organisms. In this study we evaluated the sensitivity of aquatic invertebrates to a single application of imidacloprid and an equimolar mixture of five neonicotinoids (imidacloprid, acetamiprid, thiacloprid, thiamethoxam, clothianidin) using mesocosms under Mediterranean conditions. Cyclopoida, Cloeon dipterum and Chironomini showed the highest sensitivity to neonicotinoids, with calculated NOECs below 0.2 µg/L. The sensitivity of these taxa was found to be higher than that reported in previous studies performed under less warm conditions, proving the high influence of temperature on neonicotinoid toxicity. The short-term responses of the zooplankton and the macroinvertebrate communities to similar imidacloprid and neonicotinoid mixture concentrations were very similar, suggesting that the concentration addition model can be used as a plausible hyphotesis to assess neonicotinoid mixture effects in aquatic ecosystems. Long-term mixture toxicity assessments, however, should consider the fate of the evaluated substances in the environment of concern. As part of this study, we also demonstrated that Species Sensitivity Distributions constructed with chronic laboratory toxicity data and calculated (multi-substance) Potentially Affected Fractions provide an accurate estimation to asssess the ecotoxicologial risks of imidacloprid and neonicotinoid mixtures to aquatic invertebrate species assemblages.
Morphological and molecular effects of two diluted bitumens on developing fathead minnow (Pimephales promelas) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-10 F.M. Alsaadi, B.N. Madison, R.S. Brown, P.V. Hodson, V.S. Langlois
Canada has experienced a significant increase in the transport of diluted bitumen (dilbit), a predominant oil sands product that combines bitumen with diluents derived from oil-gas condensates and other proprietary compounds. The toxicity of dilbit to fish embryos, which are immobile and thus at a high risk of exposure to oil in the event of a spill, remains largely unknown for most species. This study assessed the toxicity of water accommodated fractions (WAF) and chemically enhanced water accommodated fractions (CEWAF) of two winter dilbit blends, Access Western Blend (AWB) and Cold Lake Blend (CLB), to fathead minnow (Pimephales promelas) embryos. The TPH-F EC50 s for malformations were 834 and 1058 μg/L for AWB WAF and CEWAF, respectively, and 500 and 715 μg/L for CLB WAF and CEWAF, respectively. Levels of cyp1a mRNA increased up to 46- and 69-fold, respectively, reflecting increasing exposure to polycyclic aromatic compounds (PACs) in AWB and CLB. Similarly, levels of gst mRNA were elevated up to 3.8-fold and 2.7-fold with increasing total concentrations of PACs in AWB and CLB, respectively. However, there were no significant changes in mRNA levels of p53, sod, cat, and gsr. These results suggest that the expression of cyp1a and gst may serve as biomarkers for dilbit exposure in fathead minnow, furthering our understanding of dilbit-responsive indicators of toxicity in fish species native to North America. This study is important as it utilizes the same exposure methodology to examine the toxicity of two commonly used Canadian dilbits, facilitating comparison of dilbit toxicity.
Deepwater Horizon Oil alone and in Conjunction with Vibrio anguillarum Exposure Modulates Immune Response and Growth in Red Snapper (Lutjanus campechanus) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-08 Maria L. Rodgers, Ryan Takeshita, Robert J. Griffitt
This study examined the impacts of Macondo oil from the Deepwater Horizon oil spill, both alone and in conjunction with exposure to the known fish pathogen Vibrio anguillarum, on the expression of five immune-related gene transcripts of red snapper (il8, il10, tnfa, il1b, and igm). In order to elucidate this impact, six different test conditions were used: one Control group (No oil/No pathogen), one Low oil/No pathogen group (tPAH50 = 0.563 µg/L), one High oil/No pathogen group (tPAH50 = 17.084 µg/L, one No oil/Pathogen group, one Low oil/Pathogen group (tPAH50 = 0.736 µg/L), and one High oil/Pathogen group (tPAH50 = 15.799 µg/L). Fish were exposed to their respective oil concentrations for one week. On day 7 of the experiment, all fish were placed into new tanks (with or without V. anguillarum) for one hour. At three time points (day 8, day 10, and day 17), fish organs were harvested and placed into RNAlater, and qPCR was run for examination of the above specific immune genes as well as cyp1a1. Our results suggest that cyp1a1 transcripts were upregulated in oil-exposed groups throughout the experiment, confirming oil exposure, and that all five immune gene transcripts were upregulated on day 8, but were generally downregulated or showed no differences from controls on days 10 and 17. Finally, both oil and pathogen exposure had impacts on growth.
Characterization of Laguncularia racemosa transcriptome and molecular response to oil pollution Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-07 Fernanda Alves de Freitas Guedes, Priscilla de Barros Rossetto, Fábia Guimarães, Maurício Wolf Wilwerth, Jorge Eduardo Santos Paes, Marisa Fabiana Nicolás, Fernanda Reinert, Raquel Soares Peixoto, Márcio Alves-Ferreira
Mangroves are ecosystems of economic and ecological importance. Laguncularia racemosa (Combretaceae), popularly known as white mangrove, is a species that greatly contributes to the community structure of neotropical and West African mangrove forests. Despite the significance of these ecosystems, they have been destroyed by oil spills that can cause yellowing of leaves, increased sensitivity to other stresses and death of trees. However, the molecular response of plants to oil stress is poorly known. In this work, Illumina reads were de novo assembled into 46,944 transcripts of L. racemosa roots and leaves, including putative isoform variants. In addition to improving the genomic information available for mangroves, the L. racemosa assembled transcriptome allowed us to identify reference genes to normalize quantitative real-time PCR (qPCR) expression data from oil-stressed mangrove plants, which were used in RNASeq validation. The analysis of expression changes induced by the oil exposure revealed 310 and 286 responsive transcripts of leaves and roots, respectively, mainly up-regulated. Enriched GO categories related to chloroplasts and photosynthesis were found among both leaf and root oil-responsive transcripts, while “response to heat” and “response to hypoxia” were exclusively enriched in leaves and roots, respectively. The comparison of L. racemosa 12-h-oil-stressed leaf expression profile to previous Arabidopsis heat-stress studies and co-expression evidence also pointed to similarities between the heat and oil responses, in which the HSP-coding genes seem to play a key role. A subset of the L. racemosa oil-responsive root genes exhibited similar up-regulation profiles to their Arabidopsis homologs involved in hypoxia responses, including the HRA1 and LBD41 TF-coding genes. Genes linked to the ethylene pathway such as those coding for ERF TFs were also modulated during the L. racemosa root response to oil stress. Taken together, these results show that oil contamination affects photosynthesis, protein metabolism, hypoxia response and the ethylene pathway in L. racemosa 12-h-oil-exposed leaves and roots.
Toxic effects of nickel oxide (NiO) nanoparticles on the freshwater alga Pseudokirchneriella subcapitata Aquat. Toxicol. (IF 3.884) Pub Date : 2018-09-01 Cátia A. Sousa, Helena M.V.M. Soares, Eduardo V. Soares
Over the last decade, concerns have been raised regarding the potential health and environmental effects associated with the release of metal oxide nanoparticles (NPs) into ecosystems. In the present work, the potential hazards of nickel oxide (NiO) NPs were investigated using the ecologically relevant freshwater alga Pseudokirchneriella subcapitata. NiO NP suspensions in algal OECD medium were characterized with regard to their physicochemical properties: agglomeration, surface charge, stability (dissolution of the NPs) and abiotic reactive oxygen species (ROS) production. NiO NPs formed loose agglomerates and released Ni2+. NiO NPs presented a 72 h-EC50 of 1.6 mg L-1, which was evaluated using the algal growth inhibition assay and allowed this NP to be classified as toxic. NiO NPs caused the loss of esterase activity (metabolic activity), the bleaching of photosynthetic pigments and the intracellular accumulation of reactive oxygen species (ROS) in the absence of the disruption of plasma membrane integrity. NiO NPs also disturbed the photosynthetic process. A reduction in the photosynthetic efficiency (ΦPSII) accompanied by a decrease in the flow rate of electrons through the photosynthetic chain was also observed. The leakage of electrons from the photosynthetic chain may be the origin of the ROS found in the algal cells. The exposure to NiO NPs led to the arrest of the cell cycle prior to the first cell division (primary mitosis), an increase in cell volume and the presence of aberrant morphology in the algal cells. In this work, the use of different approaches allowed new clues related to the toxicity mechanisms of NiO NPs to be obtained. This work also contributes to the characterization of the environmental and toxicological hazards of NiO NPs and provides information on the possible adverse effects of these NPs on aquatic systems.
Effects of zinc pyrithione on biochemical parameters of the freshwater Asian clam Corbicula fluminea Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-30 Ana Filipa Nogueira, Joana Luísa Pereira, Sara Cristina Antunes, Fernando Gonçalves, Bruno Nunes
Zinc pyrithione (ZnPT) is an organometallic biocide with bactericide, algaecide, and fungicide activity. Considering this biological activity, ZnPT has been used in anti-fouling paints, and also in human therapeutics and cosmetics, in shampoos to treat dandruff and seborrhoea. Despite its potential uses and consequent presence in the aquatic environment, the ecotoxicological effects of ZnPT are poorly understood. This work aims to characterize the effects of ZnPT in biochemical parameters of the Asian clam, one of the most invasive bivalves known for its biofouling action in hydro-dependent industries, using a classical (LC50 determination) and a biomarker-based approach (quantification of the activities of catalase, GSTs, and acetylcholinesterase, and also the muscle glycogen content). The here determined LC50-96 h for zinc pyrithione was 2.17 mg/L. ZnPT caused significant increases in the activity of catalase and of cholinesterases. These findings evidence the pro-oxidative effects caused by the metabolism of ZnPT. Despite the absence of clear effects, it is important to stress that the presence of ZnPT in the wild is usually accompanied by other pyrithiones, whose co-existence can contribute to the exertion of considerable toxic effects.
Do trace metal contamination and parasitism influence the activities of the bioturbating mud shrimp Upogebia cf. pusilla? Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-28 Annabelle Dairain, Xavier de Montaudouin, Patrice Gonzalez, Aurélie Ciutat, Magalie Baudrimont, Olivier Maire, Alexia Legeay
Mud shrimp are considered as among the most influential ecosystem engineers in marine soft bottom environments because of their significant bioturbation activity and their high density. These organisms play a key role on the physical structure of sediments through intense sediment reworking activity and also deeply influence geochemical properties of sediments via frequent bioirrigation events. The influence that mud shrimp have on the environment is related to the magnitude of bioturbation processes and subsequently depends on their physiological condition. In natural environments, several factors act together and influence the well-being of organisms. Among them, the deleterious role of parasites on the physiology and the behavior of their host is well established. Aquatic organisms are also subject to pollutants released by anthropogenic activities. However, the effect of both stressors on the fitness and bioturbation activity of mud shrimp has never been investigated yet.We conducted a 14-day ex-situ experiment to evaluate the influence of trace metal contamination (cadmium Cd) and parasitism infestation on the gene expression (molecular endpoint) and sediment reworking activity (behavioral endpoint) of the mud shrimp Upogebia cf. pusilla. At completion, mud shrimp exhibited substantial Cd bioaccumulation, with parasitized organisms showing a significantly lower contaminant burden than unparasitized organisms. Cadmium contamination induces modifications of gene expression in both unparasitized and parasitized organisms. We report an antagonistic effect of both stressors on gene expression, which cannot be fully explained by lower Cd bioaccumulation. At the behaviour level, parasitism seems to reduce the sediment reworking activity of mud shrimp, while Cd contamination appears to stimulate this activity. This study highlights that the effects of multiple stressors may be quite different from the effect of each stressor considered individually. It should also motivate for more studies evaluating the influence of multiple stressors on different endpoints encompassing various levels of organization.
Comparative Transcriptome Analysis between the Short-term Stress and Long-term Adaptation of the Ruditapes philippinarum in Response to Benzo[a]pyrene Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-28 Hongdan Wang, Luqing Pan, Xu Ruiyi, Jingjing Miao, Lingjun Si, Luqing Pan
In order to monitor the pollution of polycyclic aromatic hydrocarbons (PAHs) in the seawater environment, screening biomarkers capable of monitoring PAHs is the focus of many studies. The transcriptomic profiles of the digestive gland tissue from the R. philippinarum groups after the exposure to BaP (4 μg/L) at four time points (0, 0.5, 6 and 15 days) were investigated to globally screen the key genes and pathways involved in the responses to short-term stress and long-term adaptation of BaP resistance. By comparative transcriptome analysis, 233, 282 and 58 differentially expressed genes (DEGs) were identified at 0.5 day, 6 day and 15 day (vs 0 day). The differential expression genes were related to stress response, detoxification metabolic process and innate immunity. DEGs of each group at different stages were clustered in six profiles based on gene expression pattern. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis were used on all genes to determine the biological functions and processes. We selected Multidrug resistance protein 3 (MRP3), transcriptional regulator ATRX-like isoform X2 (ATRX) as biomarker indicator genes for short-term pollution monitoring and NADH dehydrogenase [ubiquinone] 1 (NQO1), Complement C1q-like protein 4 (C1q), Glutathione-S-transferase theta (GST), E3 ubiquitin-protein ligase (E3) for long-term pollution monitoring based on the different expression patterns and the function in detoxification and antioxidant defense system. Besides, the expression of seven genes was measured through Quantitative real-time PCR (qPCR) according to their gene expression patterns which was confirmed by the DGE analysis. Taken together, adoption of transcriptomic analysis to explore the bivalves’ mRNA abundance changes and detoxification metabolic mechanism under the BaP stress at different time points can aid the development of sensitive and informed molecular endpoints for application towards ecotoxicogenomic monitoring of bivalves.
Adverse effects of two pharmaceuticals acetaminophen and oxytetracycline on life cycle parameters, oxidative stress, and defensome system in the marine rotifer Brachionus rotundiformis Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-27 Jun Chul Park, Deok-Seo Yoon, Eunjin Byeon, Jung Soo Seo, Un-Ki Hwang, Jeonghoon Han, Jae-Seong Lee
To investigate the adverse effect of two widely used pharmaceuticals, paracetamol (acetaminophen [APAP]) and oxytetracycline (OTC) on the marine rotifer Brachionus rotundiformis (B. rotundiformis), the animals were exposed to nvarious environmentally-relevant concentrations. Up to date, acetaminophen and oxytetracycline have been considered as toxic, if used above threshold concentration, i.e. overdosed. However, this study demonstrated these two pharmaceuticals even at low concentration (i.e., μg/L scale) elicited oxidative stress through the generation of reactive oxygen species (ROS) along with the increased glutathione S-transferase activity, despite no-observed effect in in-vivo population growth. To validate the adverse effects of the two pharmaceuticals at relatively low concentrations, mRNA expression analysis was performed of the entire set of gened encoding 26 cytochrome P450s (CYPs) of phase I and 19 glutathione S-transferases (GSTs) of phase II of the rotifer B. rotundiformis. The mRNA expression analysis suggested specific genes CYP3045A2 and GSTσ1, GSTσ4, and GSTω1 take part in detoxification of APAP and OTC, resulting in no significant changes in the population growth and undetermined no observed effect concentration (NOEC) in the marine rotifer B. rotundiformis.
Monitoring sublethal changes in fish physiology following exposure to a light, unweathered crude oil Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-24 Sharon E. Hook, Julie Mondon, Andrew T. Revill, Paul A. Greenfield, Sarah A. Stephenson, Joanna Strzlecki, Patricia Corbett, Emily Armstrong, Jing Song, Hai Doan, Skye Barrett
Biomarkers are frequently used to determine the exposure of fish to petroleum hydrocarbons following an oil spill. These biomarkers must be chosen carefully if they are to be used to determine sublethal toxic impacts as well as oil exposure. Many commonly used biomarkers relate to the metabolism of high molecular weight, typically pyrogenic, polycyclic aromatic hydrocarbons (PAHs), which are not abundant in unweathered crude oil. The goal of this study was to compare the efficacy of different biomarkers, including histological examination and transcriptomic profiling, in showing exposure to oil and the potential for sublethal toxic impacts. To achieve these goals, subadults/adults of the spotted dragonet (Repomucenus calcaratus) was exposed to a representative light, unweathered Australian oil for 96 h, so that the physiological changes that occur with exposure could be documented. Fish were then transferred to clean sediment for 90 h to quantify recovery. Biomarker changes, including PAH metabolites, 7-ethoxyresorufin O-deethylase (EROD), and histopathology, are presented in this work. In addition, a de novo transcriptome for the spotted dragonet was assembled, and differential transcript abundance was determined for the gill and liver of petroleum-exposed fish relative to a control. Increased levels of some biliary phenanthrene metabolites were seen throughout the exposure period. EROD levels showed modest, but not significant, increases. Transcriptomic differences were noted in the abundances of transcripts with a role in inflammation, primary metabolism and cardiac function. The patterns of transcript abundance in the gill and the liver changed in a manner that reflected exposure and recovery. The histology showed elevated prevalence of lesions, most notably vacuolization in liver and heart tissue, multi-organ necrosis, and lamellar epithelial lifting and telangiectasia in the gill. These findings suggest that short-term exposures to low molecular weight PAHs could elicit changes in the health of fish that are well predicted by the transcriptome. Furthermore, when light oil is released into the environment, exposure and subsequent risk would be better estimated using phenanthrene metabolite levels rather than EROD. This study also adds to the weight of evidence that exposure to low molecular weight PAHs may cause cardiac problems in fish. Further study is needed to determine the impact of these changes on reproductive capacity, long-term survival, and other population specific parameters.
Age-dependent survival, stress defense, and AMPK in Daphnia pulex after short-term exposure to a polystyrene nanoplastic Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-23 Zhiquan Liu, Mingqi Cai, Ping Yu, Minghai Chen, Donglei Wu, Meng Zhang, Yunlong Zhao
The widespread occurrence and accumulation of micro- and nanoplastics in aquatic environments has become a growing global concern. Generally, natural aquatic populations are characterized by a variety of multi-structured age groups, for which physiological and biochemical responses typically differ. The freshwater cladoceran, Daphnia pulex, is a model species used extensively in environmental monitoring studies and ecotoxicology testing. Here, the effects of a polystyrene nanoplastic on the physiological changes (i.e., survival) and expression levels of stress defense genes (i.e., those encoding antioxidant-mediated and heat shock proteins) in this freshwater flea were measured. Results from acute bioassays were used to determine the respective nanoplastic LC50 values for five age groups (1-, 4-, 7-, 14- and 21-day-old individuals): the obtained values for the 1- and 21-day-old D. pulex groups were similar (i.e., not significantly different). The expression levels of genes encoding key stress defense enzymes and proteins—SOD, CAT, GST, GPx, HSP70, and HSP90—were influenced by the nanoplastic in all the age groups, but not in the same way for each. Significant differences were observed among all age groups in their expression of the gene encoding the energy-sensing enzyme AMPK (adenosine monophosphate-activated protein kinase) α, β, and γ following exposure to the nanoplastic. Moreover, the expression of AMPK α was significantly increased in the 1-, 7-, and 21-day-old individuals exposed to nanoplastic relative to the control group. Together, these results indicate that age in D. pulex affects the sensitivity of its individuals to pollution from this nanoplastic, primarily via alterations to vital physiological and biochemical processes, such as cellular energy homeostasis and oxidation, which were demonstrated in vivo. We speculate that such age-related effects may extend to other nanoplastics and forms of pollution in D. pulex and perhaps similar marine organisms.
The role of GST omega in metabolism and detoxification of arsenic in clam Ruditapes philippinarum Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-23 Lizhu Chen, Huifeng Wu, Jianmin Zhao, Wei Zhang, Li Zhang, Shan Sun, Dinglong Yang, Bo Cheng, Qing Wang
The major hazard of arsenic in living organisms is increasingly being recognized. Marine mollusks are apt to accumulate high levels of arsenic, but knowledge related to arsenic detoxification in marine mollusks is still less than sufficient. In this study, arsenic bioaccumulation as well as the role of glutathione S-transferase omega (GSTΩ) in the process of detoxification were investigated in the Ruditapes philippinarum clam after waterborne exposure to As(III) or As(V) for 30 days. The results showed that the gills accumulated significantly higher arsenic levels than the digestive glands. Arsenobetaine (AsB) and dimethylarsenate (DMA) accounted for most of the arsenic found, and monomethylarsonate (MMA) can be quickly metabolized. A subcellular distribution analysis showed that most arsenic was in biologically detoxified metal fractions (including metal-rich granules and metallothionein-like proteins), indicating their important roles in protecting cells from arsenic toxicity. The relative mRNA expressions of two genes encoding GSTΩ were up-regulated after arsenic exposure, and the transcriptional responses were more sensitive to As(III) than As(V). The recombinant GSTΩs exhibited high activities at optimal conditions, especially at 37 °C and pH 4 – 5, with an As(V) concentration of 60 mM. Furthermore, the genes encoding GSTΩ significantly enhance the arsenite tolerance but not the arsenate tolerance of E. coli AW3110 (DE3) (ΔarsRBC). It can be deduced from these results that GSTΩs play an important role in arsenic detoxification in R. philippinarum.
Hexavalent chromium induces oxidative stress and mitochondria-mediated apoptosis in isolated skin fibroblasts of Indo-Pacific humpback dolphin Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-20 Xinjian Yu, Ri-qing Yu, Duan Gui, Xiyang Zhang, Fenping Zhan, Xian Sun, Yuping Wu
The increasing gas emissions and industrial wastewater discharge of anthropogenic hexavalent chromium (Cr(VI)) have been growing health concerns to the high trophic level marine mammals. Our previous studies showed that Indo-Pacific humpback dolphin (Sousa chinensis), stranded on the Pearl River Estuary region, contained exceedingly high levels of Cr in their skin-tissue. Unfortunately, the molecular toxic mechanisms on this mammal are absent, limiting our understanding of the eco-physiological impacts of Cr(VI) on dolphins. Thus, the cytotoxicity effects of Cr(VI) were analyzed on fibroblasts we isolated from the skin of S. chinensis (ScSF). This study showed that Cr(VI) markedly inhibited the viability of ScSF cells via induction of apoptosis accompanied by an increase in the production of reactive oxygen species and the population of G2/M arrest or apoptotic sub-G1 phase cells, up-regulation of p53, and activation of caspase-3. Further investigation on intracellular mechanisms indicated that Cr(VI) induced depletion of mitochondrial membrane potential in cells through regulating the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins, resulting in decrease of the ATP level, cytochrome c release from mitochondria into cytosol, and the activation of caspase-9. Furthermore, antioxidants N-acetylcysteine and vitamin C displayed chemoprotective activity against Cr(VI) via suppression of p53 expression, indicating that the Cr(VI)-induced cell death may be mediated by oxidative stress. Overall, these results provide insights into the potential mechanisms underlying the cytotoxicity of Cr(VI) in Indo-Pacific humpback dolphin skin cells, offer experimental support for the proposed protective role of antioxidants in Cr(VI)-induced toxicity, and suggest that Cr(VI) contamination is one of key health concern issues for the protection of Indo-Pacific humpback dolphin.
Blinded by the light: increased chlorophyll fluorescence of herbicide-exposed periphyton masks unfavorable structural responses during exposure and recovery Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-19 Alexander Feckler, Jelena Rakovic, Maria Kahlert, Rikard Tröger, Mirco Bundschuh
In surface waters within agricultural catchments, periphyton – i.e., biofilms containing algae, heterotrophs, and associated detritus – is subjected to multiple stressors including herbicides. Although herbicide effects on periphyton are frequently studied, the focus has been on photosynthesis-inhibiting herbicides while other modes of toxic action have received little attention. Against this background, a 21-days-lasting bioassay was conducted, during which mature periphytic communities were exposed to the carotenoid-biosynthesis-inhibiting herbicide diflufenican for 12 days (up to 10 µg/L; n = 4), followed by a 9-days-lasting recovery phase in herbicide-free medium. Variables related to periphytic functioning (photosynthetic efficiency and non-photochemical quenching) and structure (pigment concentrations, biomass, and algal community structure) were quantified every third day during both experimental phases. Exposure to ≥ 0.2 µg diflufenican/L resulted in 20-25% and 25-30% lowered carotenoid and chlorophyll a concentrations, respectively, likely explained by a reduced algal biovolume as well as diflufenican’s mode of toxic action and thus a shift towards a higher heterotrophy of the communities. Despite these adverse effects on the photosynthetic apparatus, the photosynthetic efficiency increased by up to ~15% under diflufenican exposure judged on higher chlorophyll fluorescence. This may be explained by an up to ~60% reduced non-photochemical quenching as well as binding of diflufenican to the pigment-protein membrane complex of the photosystem II, two processes causing higher chlorophyll fluorescence. Additionally, phototrophs may have actively increased energy assimilation to cope with higher energy demands under chemical stress. Although periphyton showed some recovery potential following the exposure phase, observed as increasing chlorophyll a concentrations and non-photochemical quenching, periphyton may not be able to quickly recover from stress given the persistent increase in the photosynthetic efficiency. While the processes underlying the observed effects yet remain speculative, the results suggest a shift towards a higher degree of heterotrophy in periphytic communities ultimately increasing the importance of heterotrophic ecosystem functions at impacted sites over the long term.
Growth, energy metabolism and transcriptomic responses in Chinese mitten crab (Eriocheir sinensis) to benzo[α]pyrene (BaP) toxicity Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-18 Na Yu, Qingqing Ding, Erchao Li, Jian G. Qin, Liqiao Chen, Xiaodan Wang
Benzo(a)pyrene (BaP) is a highly toxic polycyclic aromatic hydrocarbon and has strong affinity to suspended materials and sediments in the aquatic environment. Most crustaceans are benthic species and are easily affected by the pollution in the sediments, but there is little information on the response mechanism of crustaceans to BaP exposure. This study compared the growth and hepatopancreas transcriptomic responses of the Chinse mitten crab (Eriocheir sinensis) exposed to 0, 0.15 (BaP1) and 0.45 μg /L (BaP2) for 28 days. Crab survival and weight gain were reduced in the water born BaP in a dose-dependent way. The contents of hepatopancreas glycogen, triglyceride, total amino acids and lactic acid were all decreased after BaP exposure, indicating possible more energy consumption during detoxification. In the transcriptome analysis, a total of 106.65 million clean reads were obtained and assembled into 81,714 unigenes with an average length of 594 bp and N50 of 808 bp. Under 0.15 or 0.45 μg /L BaP exposure, 922 and 1129 unigenes in crabs were significantly expressed, annotated to 676 and 802 Gene Ontology (GO) terms respectively. The “cellular process” was the leading category for both concentrations. Thirteen significantly changed pathways were identified in both Control vs BaP1 and Control vs BaP2 groups. These pathways were divided into four different parts according to their reported functions, including metabolism, environmental information processing, organismal systems and cellular processes. Nice out of thirteen pathways in BaP1 were related to metabolism, containing amino acid metabolism, phenylpropanoid biosynthesis, monobactam biosynthesis and styrene degradation. Almost all the pathways related with the biosynthesis processes were down-regulated, while the degradation pathways were up-regulated. Seven out of thirteen pathways were classified into metabolism category in BaP2. These pathways were mostly associated with stress resistance rather than supplying energy. This study indicates that both concentrations of BaP disturbed nutrient metabolism, immune response and defense system in the crabs, while exposure to a higher concentration had a greater impact on immunity system than on metabolism. This study provides a better understanding of the underlying molecular and regulatory mechanisms in crustaceans coping with BaP toxicity.
Social competition in red drum (Sciaenops ocellatus) is influenced by crude oil exposure Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-18 Alexis J. Khursigara, Jacob L. Johansen, Andrew J. Esbaugh
The present study examined impacts of crude oil exposure on dyad competition in juvenile red drum. Following the 2010 Deepwater Horizon oil spill, it has become well established that oil exposure can constrain maximum metabolic rate, reduce aerobic scope and exercise performance in marine fish. Aerobic scope is one of the physiological characteristics that is a known determinant of dominance in fish social hierarchy formation. As such, oil exposure may predispose individuals to subordinate social status, complete with the concomitant ecological costs. We tested this hypothesis on the gregarious Gulf of Mexico species, the red drum (Sciaenops ocellatus). Using a standard dyad, one-on-one, test design, we first assessed the parameters – including size and aerobic scope– that predict social dominance. Of the tested parameters, only aerobic scope was predictive of social dominance, with dominant individuals consistently having higher aerobic scopes than subordinates. Hierarchy formation between individuals exposed to one of two oil concentrations (5.7 ± 0.5 and 9.0 ± 0.2 µg l-1 ΣPAH50) and unexposed conspecifics were then investigated. As hypothesized, fish exposed to both oil concentrations were more likely to be subordinate than what would occur by random chance. These results demonstrate that the physiological constraints imposed by oil exposure can affect social status and behavior in fishes, which can have downstream consequences for ecological fitness.
UV-B radiation induces DEHP degradation and their combined toxicological effects on Scenedesmus acuminatus Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-16 Chao Zhang, Mu Xie, Jing Chen, Yurui Zhang, Sijie Wei, Xinyue Ma, Ling Xiao, Lanzhou Chen
The co-contamination discharge of Phthalate esters (PAEs) by human activities and the increased UV radiation is increasing in aquatic ecosystems. However, little information is available about the combined detrimental effects of UV and PAEs on phytoplankton. In this study, the combined effects of UV-B irradiation and di-(2-ethylhexyl) phthalate (DEHP) on photosynthesis and antioxidant system of Scenedesmus acuminatus, and the DEHP degradation were investigated. Results showed that UV-B radiation decreased the chlorophyll a fluorescence yield, photosynthetic activity (Fv/Fm), pigment content and superoxide dismutase activity. This radiation also increased the reactive oxygen species (ROS) production and soluble protein and malondialdehyde contents. UV-B radiation with 10 mg L-1 DEHP improved the Fv/Fm and alleviated the cell damage of S. acuminatus, and the addition of high DEHP concentration (≥50 mg L-1) aggravated cell damage. The ROS generation also decreased with the increased DEHP concentration. UV-B radiation can effectively promote the DEHP degradation, with the highest degradation rate of 89.9% at an initial DEHP concentration of 10 mg L-1 within 6 h. This result may be attributed to that UV-B irradiance induced DEHP degradation under the regulation of ROS generated by S. acuminatus. Our findings will contribute to the understanding of the combined toxic mechanisms of UV-B and DEHP and in the evaluation of ecological environment risks for primary producers in aquatic ecosystems.
Environmental Risks of ZnO Nanoparticle Exposure on Microcystis aeruginosa: Toxic Effects and Environmental Feedback Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-16 Yulin Tang, Huaijia Xin, Shu Yang, Meiting Guo, Tyler Malkoske, Daqiang Yin, Shengji Xia
The vast majority of studies measure the toxic effect of organisms exposed to nanoparticles (NPs) while there is still a lack of knowledge about the influence of NPs on the aquatic environment. It is unknown whether or not the interaction between NPs and algae will result in the variation of algal organic matter (AOM) and stimulate the production of more algal toxins. In this study, zinc oxide nanoparticles (nano-ZnO) as a typical representative of metal oxide NPs were used to evaluate the toxic effects and environmental feedback of Microcystis aeruginosa. Reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to explain the toxicity mechanism. Changes of AOM, including the production of toxins, the molecular weight distribution and the excitation-emission matrices of algal solution were also studied as environmental feedback indicators after nano-ZnO destroyed the algae. As the nano-ZnO exceeded the comparable critical concentration (1.0 mg/L), the algae were destroyed and intracellular organic matters were released into the aquatic environment, which stimulated the generation of microcystin-LR (MC-LR). However, it is worth noting that the concentration of nano-ZnO would need to be high (at mg/L range) to stimulate more MC-LR production. These findings are expected to be beneficial in interpreting the toxicity and risks of the releasing of NPs through the feedback between algae and the aquatic environment.
Oxidative stress responses and cellular energy allocation changes in microalgae following exposure to widely used human antibiotics Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-15 Adeolu O. Aderemi, Sara C. Novais, Marco F. Lemos, Luís M. Alves, Colin Hunter, Ole Pahl
The individual effect of four human antibiotics on the microalgae Raphidocelis subcapitata was investigated following a 120-h exposure. The effects were assessed by analyzing growth, and biochemical parameters related with: 1) antioxidant capacity and oxidative damage by measuring superoxide dismutase (SOD) activity and lipid peroxidation (LPO) levels; and 2) cellular energy allocation (CEA) by quantifying the content in energy reserves, which represents the energy available (Ea), and the electron transport system activity that represents a measure of oxygen and cellular energy consumption (Ec). Growth yield inhibitory concentrations of sulfamethoxazole (18-30%), clarithromycin (28.7%), ciprofloxacin (28%) and erythromycin (17-39%) were found to elicit a considerable increase in Ec, thereby causing a significant decrease in the CEA. The elevated Ec can be a result of the need to respond to oxidative stress occurring under those conditions given the significant increase in SOD activity at these levels. For sulfamethoxazole, erythromycin and ciprofloxacin, the antioxidant responses do not seem to be enough to cope with the reactive oxygen species and prevent oxidative damage, given the elevated LPO levels observed. A stimulatory effect on growth yield was observed (up to 16%) at ciprofloxacin lowest concentration, which highly correlated with the increase in CEA. Based on the no observed effect concentration (NOECs) and/or effective concentration (EC10) results, Ec, SOD and CEA were more sensitive than the classical endpoint of growth rate for all the tested antibiotics. By revealing the antibiotic stress effects in R. subcapitata at the cellular level, this study suggests CEA as a more reliable indicator of the organisms’ physiological status.
Testing wastewater treatment plant effluent effects on microbial and detritivore performance: A combined field and laboratory experiment Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-14 Libe Solagaistua, Ioar de Guzmán, Miren Barrado, Leire Mijangos, Nestor Etxebarria, Gonzalo García-Baquero, Aitor Larrañaga, Daniel von Schiller, Arturo Elosegi
Impact of multiple stressors on biomarker responses in sympatric dreissenid populations Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-11 Marine Potet, Laure Giambérini, Sandrine Pain-Devin, Audrey Catteau, Danièle Pauly, Simon Devin
Dreissenid mussels, well-known invaders of the northern hemisphere, also constitute good biomonitors for freshwater quality assessment. Whereas the oldest, Dreissena polymorpha, is relatively well-known, the new invasive, Dreissena rostriformis bugensis, has been very little characterized. The aim of this study was to compare subcellular biomarker responses within these species, by taking into account inter- and intra-specific variability in biomarker responses during a multi-stress assessment. Indeed, due to local environmental conditions and genetic background, biomarker responses can be different between populations of a given species, not only between species. To evaluate these differential responses, we sampled mussels on two sites where they are both present in sympatry (plus another D. r. bugensis population), and we exposed these populations in laboratory to different stressors: thermal stress (12 or 17 °C), dietary stress (fed or unfed), contamination (nickel at 0, 20 and 500 µg.L-1) and exposure duration (0, 4 or 8 days). Results mainly evidenced strong inter-species and inter-population differences, underlining the need to discriminate correctly between the two species and to know well the populations used in biomonitoring. Results also evidenced thermal and food stress-related effects. The numerous data obtained during this multi-stress experiment also highlight the complexity of working on several stressors and analysing the associated results.
Effects of nanosilver on Mytilus galloprovincialis hemocytes and early embryo development Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-07 M. Auguste, C. Ciacci, T. Balbi, A. Brunelli, V. Caratto, A. Marcomini, R. Cuppini, L. Canesi
Silver nanoparticles (AgNP), one of the main nanomaterials for production and use, are expected to reach the aquatic environment, representing a potential threat to aquatic organisms. In this study, the effects of bare AgNPs (47 nm) on the marine mussel Mytilus galloprovincialis were evaluated at the cellular and whole organism level utilizing both immune cells (hemocytes) and developing embryos. The effects were compared with those of ionic Ag+(AgNO3). In vitro short-term exposure (30 min) of hemocytes to AgNPs induced small lysosomal membrane destabilization (LMS EC50 = 273.1 μg/mL) and did not affect other immune parameters (phagocytosis and ROS production). Responses were little affected by hemolymph serum (HS) as exposure medium in comparison to ASW. However, AgNPs significantly affected mitochondrial membrane potential and actin cytoskeleton at lower concentrations. AgNO3 showed much higher toxicity, with an EC50 = 1.23 μg/mL for LMS, decreased phagocytosis and induced mitochondrial and cytoskeletal damage at similar concentrations. Both AgNPs and AgNO3 significantly affected Mytilus embryo development, with EC50 = 23.7 and 1 µg/L, respectively. AgNPs caused malformations and developmental delay, but no mortality, whereas AgNO3 mainly induced shell malformations followed by developmental arrest or death. Overall, the results indicate little toxicity of AgNPs compared with AgNO3; moreover, the mechanisms of action of AgNP appeared to be distinct from those of Ag+. The results indicate little contribution of released Ag+ in our experimental conditions. These data provide a further insight into potential impact of AgNPs in marine invertebrates.
[omim][BF4]-mediated toxicity in mussel hemocytes includes its interaction with cellular membrane proteins Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-06 Vasiliki Tsarpali, Stefanos Dailianis
The current study is based on the increasing demand for the assessment of ionic liquid (IL)-mediated aquatic toxicity. Specifically, although a lot of studies have been performed so far, investigating IL-mediated adverse effects on numerous aquatic organisms, little is known about their mode of action. Given that the use of in vitro models is considered as a reliable tool for determining the mediated biological effects, the modulation of specific biochemical pathways and the onset of various forms of damage with great precision and reproducibility, mixed primary cultures of mussel Mytilus galloprovincialis hemocytes were used for investigating whether 1-octyl-3-methylimidazolium tetrafluoroborate ([omim][BF4]) mediated toxicity is related to its interaction with cellular membrane proteins. Specifically, [omim][BF4]-mediated cytotoxic, oxidative and genotoxic effects were investigated in mussel hemocytes before and after pre-treatment of cells with non-toxic concentration of guanidine hydrochloride (1 mM GndHCl). The results showed that [omim][BF4] at concentrations ranging from 0.7 to 1.75 μM can induce cytotoxic (almost <50% reduction of cell viability), oxidative (increased levels of O2•- production and lipid peroxidation by-products) and genotoxic (increased levels of DNA damage) effects, while cells pre-treated with 1 mM GndHCl showed a significant attenuation of IL’s toxic potency in all cases. According to the latter, the current study showed that [omim][BF4]-mediated toxicity could be related not only to its well-known interaction with membrane lipid bilayers, but also to its interference with membrane proteins. Using GndHCl, a chaotropic agent that disrupts the hydrogen bonding network and the stability of membrane proteins via its interference with the intramolecular interactions mediated by non-covalent forces on cellular membranes, it was firstly shown that altering the membrane integrity as well as the native state of cellular membrane proteins, by weakening the hydrophobic effect, could attenuate the possible interaction of [omim][BF4] with cellular membranes and the concomitant induction of protein-based intracellular processes, commonly linked with the induction of severe cellular damage.
Developmental neurotoxicity of triphenyl phosphate in zebrafish larvae Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-03 Qipeng Shi, Min Wang, Fengqiong Shi, Lihua Yang, Yongyong Guo, Chenglian Feng, Jingfu Liu, Bingsheng Zhou
Triphenyl phosphate (TPhP), a typical organophosphate ester, is frequently detected in the environment and biota samples. It has been implicated as a neurotoxin as its structure is similar to neurotoxic organophosphate pesticides. The purpose of the present study was to investigate its potential developmental neurotoxicity in fish by using zebrafish larvae as a model. Zebrafish (Danio rerio) embryos were exposed to 0.8, 4, 20 and 100 μg/L of TPhP from 2 until 144 h post-fertilization. TPhP was found to have high bioconcentrations in zebrafish larvae after exposure. Further, it significantly reduced locomotor activity as well as the heart rate at the 100 μg/L concentration. TPhP exposure significantly altered the content of the neurotransmitters γ-aminobutyric and histamine. Downregulation of the genes related to central nervous system development (e.g., α1-tubulin, mbp, syn2a, shha, and elavl3) as well as the corresponding proteins (e.g., α1-tubulin, mbp, and syn2a) was observed, but the gap-43 protein was found to upregulated. Finally, marked inhibition of total acetylcholinesterase activity, which is considered as a biomarker of neurotoxicant exposure, was also observed in the larvae. Our results indicate that exposure to environmentally relevant concentrations of TPhP can affect different parameters related to center nervous system development, and thus contribute to developmental neurotoxicity in early developing zebrafish larvae.
Effects of copper on hemocyte parameters in the estuarine oyster Crassostrea rivularis under low pH conditions Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-02 Xizhi Huang, Xiaoyu Jiang, Meng Sun, Sam Dupont, Wei Huang, Menghong Hu, Qiongzhen Li, Youji Wang
CREB element is essential for unfolded protein response (UPR) mediating the Cu-induced changes of hepatic lipogenic metabolism in Chinese yellow catfish (Pelteobagrus fulvidraco) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-08-01 Yu-Feng Song, Yi-Huan Xu, Mei-Qin Zhuo, Kun Wu, Zhi Luo
The present study was conducted to explore the underlying mechanism of unfolded protein response (UPR) mediating the Cu-induced changes of hepatic lipogenic metabolism in a low vertebrate, freshwater teleost yellow catfish Pelteobagrus fulvidraco. To this end, three experiments were conducted. In Exp. 1, we cloned the regions of grp78, perk, ire-1α and atf-6α promoters, and found that multiple cAMP-response element binding protein (CREB) binding sites were identified in their promoter regions. Furthermore, these CREB binding sites played crucial role in transcriptional regulation of UPR. In Exp. 2, the involvement of perk, ire-1α and atf-6α in Cu-induced changes of hepatic lipid metabolism was confirmed by specific miRNA. In Exp. 3, the regulatory mechanism of CREB underlying UPR mediating Cu-induced hepatic lipogenic metabolism were investigated. Cu induced UPR via the activation of CREB binding sites in the promoter regions of grp78, perk, ire-1α and atf-6α. In addition, the inhibition of CREB markedly attenuated the Cu-induced up-regulation of hepatic lipogenic metabolism in hepatocytes. This conclusion was further supported by the results from the trial of CREB over-expression. Taken together, the present study indicated that CREB was essential for UPR mediating Cu-induced lipogenic metabolism, supporting a mechanistic link among CREB, UPR and Cu-induced changes of lipid metabolism.
Sex-specific immunomodulatory action of the environmental estrogen 17α-ethynylestradiol alongside with reproductive impairment in fish Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-31 Roy R. Ye, Drew R. Peterson, Shin-Ichi Kitamura, Helmut Segner, Frauke Seemann, Doris W.T. Au
Estrogenic endocrine disrupting chemicals (EEDCs) are present ubiquitously in sediments and aquatic ecosystems worldwide. The detrimental impact of EEDCs on the reproduction of wildlife is widely recognized. Increasing evidence shows the immunosuppressive effects of EEDCs in vertebrates. Yet, no studies have considered concomitantly EEDC-induced impacts on reproductive impairment and immune suppression in vivo, which are deemed essential for risk assessment and environmental monitoring. In this study, EE2 was used as a representative EEDC, for parallel evaluation of EEDC-induced immune suppression (immune marker gene expression, leukocyte numbers, host resistance assay, and immune competence index) and reproductive impairment (estrogen responsive gene expression, fecundity, fertilization success, hatching success, and reproductive competence index) in an established fish model (marine medaka Oryzias melastigma), considering sex-specific induction and adaptation and recovery responses under different EE2 exposure scenarios. The findings in marine medaka reveal distinct sex differences in the EE2-mediated biological responses. For female fish, low concentration of exogenous EE2 (33 ng/L) could induce hormesis (immune enhancement), enable adaptation (restored reproduction) and even boost fish resistance to bacterial challenge after abatement of EE2. However, a prolonged exposure to high levels of EE2 (113 ng/L) not only impaired F0 immune function, but also perturbed females recovering from reproductive impairment, resulting in a persistent impact on the F1 generation output. Thus, for female fish, the exposure concentration of EE2 is more critical than the dose of EE2 in determining the impacts of EE2 on immune function and reproduction. Conversely, male fish are far more sensitive than females to the presence of low levels of exogenous EE2 in water and the EE2-mediated biological impacts are clearly dose-dependent. It is also evident in male fish that direct contact of EE2 is essential to sustain impairments of immune competence and reproductive output as well as deregulation of immune function genes in vivo. The immunomodulatory pathways altered by EE2 were deciphered for male and female fish, separately. Downregulation of hepatic tlr3 and c3 (in female) and tlr3, tlr5 and c3 (in male) may be indicative of impaired fish immune competence. Taken together, impaired immune competence in the EE2-exposed fish poses an immediate thread on the survival of F0 population. Impaired reproduction in the EE2-exposed fish can directly affect F1 output. Parallel evaluation of immune competence and reproduction are important considerations when assessing the risk of sublethal levels of EE2/EEDCs in aquatic environments.
Copper and ocean acidification interact to lower maternal investment, but have little effect on adult physiology of the Sydney rock oyster Saccostrea glomerata Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-30 Elliot Scanes, Laura M. Parker, Wayne A. O’Connor, Mitchell Gibbs, Pauline M. Ross
It remains unknown how molluscs will respond to oceans which are increasingly predicted to be warmer, more acidic, and heavily polluted. Ocean acidification and trace metals will likely interact to increase the energy demands of marine organisms, especially oysters. This study tested the interactive effect of exposure to elevated pCO2 and copper on the energetic demands of the Sydney rock oyster (Saccostrea glomerata) during reproductive conditioning and determined whether there were any positive or negative effects on their offspring. Oysters were exposed to elevated pCO2 (1000 µatm) and elevated copper (Cu 50 µg L-1 [0.787 µM]) in an orthogonal design for eight weeks during reproductive conditioning. After eight weeks, energetic demands on oysters were measured including standard metabolic rate (SMR), nitrogen excretion, molar oxygen to nitrogen (O:N) ratio, and pHe of adult oysters as well as the size and total lipid content of their eggs. To determine egg viability, the gametes were collected and fertilised from adult oysters, the percentage of embryos that had reached the trochophore stage after 24 hours was recorded. Elevated pCO2 caused a lower extracellular pH and there was a greater O:N ratio in adult oysters exposed to copper. While the two stressors did not interact to cause significant effects on adult physiology, they did interact to reduce the size and lipid content of eggs indicating that energy demand on adult oysters was greater when both elevated pCO2 and copper were combined. Despite the lower energy, there were no negative effects on early embryonic development. In conclusion, elevated pCO2 can interact with metals and cause greater energetic demands on oysters; in response oysters may lower maternal investment to offspring.
Impacts of the combined exposure to seawater acidification and arsenic on the proteome of Crassostrea angulata and Crassostrea gigas Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-30 Anthony Moreira, Etelvina Figueira, Nélia C. Mestre, Denise Schrama, Amadeu Soares, Rosa Freitas, Maria João Bebianno
Proteomic analysis was performed to compare the effects of Arsenic (As), seawater acidification (Low pH) and the combination of both stressors (Low pH + As) on Crassostrea angulata and Crassostrea gigas juveniles in the context of global environmental change. This study aimed to elucidate if two closely related Crassostrea species respond similarly to these environmental stressors, considering both single and combined exposures, to infer if the simultaneous exposure to both stressors induced a differentiated response. Identification of the most important differentially expressed proteins between conditions revealed marked differences in the response of each species towards single and combined exposures, evidencing species-related differences towards each experimental condition. Moreover, protein alterations observed in the combined exposure (Low pH + As) were substantially different from those observed in single exposures. Identified proteins and their putative biological functions revealed an array of modes of action in each condition. Among the most important, those involved in cellular structure (Actin, Atlastin, Severin, Gelsolin, Coronin) and extracellular matrix modulation (Ependymin, Tight junction ZO-1, Neprilysin) were strongly regulated, although in different exposure conditions and species. Data also revealed differences regarding metabolic modulation capacity (ATP β, Enolase, Aconitate hydratase) and oxidative stress response (Aldehyde dehydrogenase, Lactoylglutathione, Retinal dehydrogenase) of the species, which also depended on single or combined exposures, illustrating a different response capacity of both oyster species to the presence of multiple stressors. Interestingly, alterations of piRNA abundance in C. angulata suggested genome reconfiguration in response to multiple stressors, likely an important mode of action related to adaptive evolution mechanisms previously unknown to oyster species, which requires further investigation. Our findings provide a deeper insight into the complexity of C. angulata and C. gigas responses to environmental stress at the proteome level, evidencing different capacities to endure abiotic changes, with relevance regarding the ecophysiological fitness of the species and competitive advantages in a changing environment.
In vivo effects of 17α-ethinylestradiol, 17β-estradiol and 4-nonylphenol on insulin-like growth-factor binding proteins (igfbps) in Atlantic salmon Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-25 Jason P. Breves, Tara A. Duffy, Ingibjörg E. Einarsdottir, Björn Thrandur Björnsson, Stephen D. McCormick
Feminizing endocrine disrupting compounds (EDCs) affect the growth and development of teleost fishes. The major regulator of growth performance, the growth hormone (Gh)/insulin-like growth-factor (Igf) system, is sensitive to estrogenic compounds and mediates certain physiological and potentially behavioral consequences of EDC exposure. Igf binding proteins (Igfbps) are key modulators of Igf activity, but their alteration by EDCs has not been examined. We investigated two life-stages (fry and smolts) of Atlantic salmon (Salmo salar), and characterized how the Gh/Igf/Igfbp system responded to waterborne 17α-ethinylestradiol (EE2), 17β-estradiol (E2) and 4-nonylphenol (NP). Fry exposed to EE2 and NP for 21 days had increased hepatic vitellogenin (vtg) mRNA levels while hepatic estrogen receptor α (erα), gh receptor (ghr), igf1 and igf2 mRNA levels were decreased. NP-exposed fry had reduced body mass and total length compared to controls. EE2 and NP reduced hepatic igfbp1b1, -2a, -2b1, -4, -5b2 and -6b1, and stimulated igfbp5a. In smolts, hepatic vtg mRNA levels were induced following 4-day exposures to all three EDCs, while erα only responded to EE2 and E2. EDC exposures did not affect body mass or fork length; however, EE2 diminished plasma Gh and Igf1 levels in parallel with reductions in hepatic ghr and igf1. In smolts, EE2 and E2 diminished hepatic igfbp1b1, -4 and -6b1, and stimulated igfbp5a. There were no signs of compromised ionoregulation in smolts, as indicated by unchanged branchial ion pump/transporter mRNA levels. We conclude that hepatic igfbps respond (directly and/or indirectly) to environmental estrogens during two key life-stages of Atlantic salmon, and thus may modulate the growth and development of exposed individuals.
Establishment of Pantropic Spotted Dolphin (Stenella attenuata) Fibroblast Cell Line and Potential Influence of Polybrominated Diphenyl Ethers (PBDEs) on Cytokines Response Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-25 Imran Rashid Rajput, Ziyang Xiao, Sun Yajing, Summra Yaqoob, Edmond Sanganyado, Huang Ying, Yu Fei, Wenhua Liu
The presence of polybrominated diphenyl ethers (PBDEs) in the aquatic environment is an issue of major concern which may be a cause of increasing prevalence and severity of diseases in marine mammals. Although, cell culture model development and in vitro investigation approach is a prime need of time to progress immunotoxic research on aquatic mammals. In this study, we stablished fibroblast cell line (pantropic spotted dolphin) to assess the potential effects of PBDEs on cytokines response. Cells were grown in 6 well cell culture plate and complete media (DMEM and Ham's F12 nutrient mixture, fetal bovine serum, antibiotic and essential amino acids) was provided. The primary culture of (PSP-LWH) cells identification was achieved by vimentin (gene and protein) expressions. Karyotyping revealed pantropic spotted dolphin chromosomes 20 pairs with XX. Transfection was achieved by SV40 LT antigen and transfected cells were expended for passages. Stability of cell line was confirmed at various passages intervals using RT-PCR, western blotting and immunofluorescence methods. After confirmation, cell line was exposed to BDE-47 (250 ng/ml), BDE-100 (250 ng/ml) and BDE-209 (1000 ng/ml), with control group (PBS), positive control DMSO (0.1%) and negative control LPS (500 ng/ml) for 24 hr. The ELISA results showed significant increase in IL-6 in BDE- 100 and BDE-209 while IL-1β and IL-8 were found higher in BDE-47 and BDE-100. TNFα and IL-10 secretion was noted higher in control and positive control groups. Altogether, these results emphasize importance of transfected (PSP-LWHT) cell line in aquatic research and potential effects of PBDEs on fibroblast provides evident to understand immune modulating effects of PBDEs in marine mammals. The impact of PBDEs on dolphin’s fibroblast cells immune response and altered cytokine response have been presented for the first time.
Triclosan toxicity alters behavioral and hematological parameters and vital antioxidant and neurological enzymes in Pangasianodon hypophthalmus (Sauvage, 1878) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-20 Vikas Kumar Sahu, Sutanu Karmakar, Saurav Kumar, S.P. Shukla, Kundan Kumar
Triclosan and its metabolites are detected in a diverse aquatic environment and are major concerns for various aquatic organisms. The present study investigated the impact of acute and sub-lethal exposure of triclosan on behaviour, activities of acetylcholinesterase and selected antioxidant enzymes, haematological and serum gas-electrolyte parameters of Pangasianodon hypophthalmus. The 96 h LC50 of triclosan for P. hypophthalmus was estimated as 1458 μg L−1. Further, sub-lethal triclosan exposure to 1/15th (97 μg L-1), 1/10th (145 μg L-1) and 1/5th (291 μg L-1) of 96 h LC50 concentration for a period of 45 days lead to decrease in total erythrocyte count, haemoglobin content and packed cell volume of blood while total leukocyte count increased significantly (p < 0.05) as compared to control. A concentration-dependent increase in the serum chloride and decrease in partial pressure of oxygen in blood serum was noted on 45th day. An increased activity of catalase and superoxide dismutase in gill and liver tissues and inhibition of acetylcholinesterase activity in brain was observed on 15th, 30th and 45th day of exposure which was dependent on both - concentration of triclosan and duration of exposure. A significant high activity of glutathione-S-transferase in gill and liver tissue was observed in triclosan exposed groups in comparison to control during the experimental period. The study shows that long-term sub-lethal exposure of triclosan to fish can lead to several physiological alterations such as enzymatic scavenging of oxygen radicals and the normal neurological functions mediated by the enzyme acetylcholinesterase. With increasing anthropogenic activity, the study provides a convincing evidence for the necessity of a regulated use and safer disposal of triclosan to the environment.
COMPARATIVE TOXICITY OF COREXIT® 9500, OIL, AND A COREXIT®/OIL MIXTURE ON THE EASTERN OYSTER, CRASSOSTREA VIRGINICA (Gmelin) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-20 Lindsay Jasperse, Milton Levin, Katherine Tsantiris, Roxanna Smolowitz, Christopher Perkins, J. Evan Ward, Sylvain De Guise
Given their particle feeding behavior, sessile behavior, and abundance in coastal zones, bivalves are at significant risk for exposure to oil and oil dispersant following environmental disasters like the Deepwater Horizon oil spill. However, the effects of oil combined with oil dispersants on the health of oysters are not well studied. Therefore, eastern oysters (Crassostrea virginica) were exposed in vivo to Corexit® 9500, crude oil (high-energy water accommodated fraction; HEWAF), and a Corexit/oil mixture (chemically-enhanced water accommodated fraction; CEWAF) to evaluate potential toxic effects on immunological (phagocytosis and respiratory burst), physiological (feeding rate), and histological endpoints. Phagocytosis was significantly increased following CEWAF exposure only. Respiratory burst was significantly decreased following Corexit® exposure, but significantly increased following exposure to the highest concentration of CEWAF. Oyster feeding rates were significantly decreased following exposure to Corexit®, HEWAF, and CEWAF, and were most sensitive to CEWAF exposure. These modulations of important immunological and physiological functions could result in serious health outcomes for oysters, such as increased parasitism and decreased growth. Our experiments showed that subtle, sub-lethal effects occurred following acute in vivo exposure to Corexit®, HEWAF, and CEWAF, though oysters were not equally sensitive to the three components. Data from this study can be used for more accurate risk assessment concerning the impact of oil and Corexit® on the health of oysters.
Gonadal differentiation and its sensitivity to androgens during development of Pelophylax nigromaculatus Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-20 Yuan-Yuan Li, Tan Meng, Kun Gao, Zhan-Fen Qin
Our previous observations proposed Pelophylax nigromaculatus as a model species for studying the masculinizing effects of androgenic EDCs in amphibians. To better develop this model species, we studied the process of the gonadal differentiation/development and the sensitive stage to androgens. We found that the earliest sexual dimorphism in gonads at morphological and histological levels occurred at stages 38-40 and stage 36 respectively. Further examination of molecular markers for testicular and ovarian differentiation during development revealed that the cyp17 and cyp19 expressions were sexually dimorphic from stage 32 and stage 36 respectively. Further, we investigated the sex-reversal induced by 100 ng/L 5α-dihydrotestosterone (DHT) when exposures were initiated at stages 24, 26 and 28. We found that when exposed from stage 24, DHT resulted masculinization of all tadpoles with no typical ovaries, whereas exposures from stage 26 or 28 dramatically reduced the effect of DHT. Our findings show that gonads of P. nigromaculatus are bipotential at stage 24, in the process of differentiation at stage 26 and determined to become either testis or ovary at stage 28. Altogether, exposure of P. nigromaculatus should begin at stage 24 in order to sensitively detect masculinizing effects of EDCs. Present study provides useful information about the gonadal differentiation and development in P. nigromaculatus for effectively evaluating masculinizing effects of EDCs on gonads.
Adverse effects of the insecticides chlordecone and fipronil on population growth and expression of the entire cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and the marine rotifer Brachionus plicatilis Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-19 Young Hwan Lee, Jun Chul Park, Un-Ki Hwang, Jae-Seong Lee, Jeonghoon Han
Chlordecone and fipronil are used as an insecticide and have been widely detected in the aquatic environments. However, their toxicity is still poorly investigated in aquatic invertebrates. In this study, we examined effects of chlordecone and fipronil on population growth and transcriptional regulation of the entire cytochrome P450 (CYP) genes in the freshwater rotifer Brachionus calyciflorus and the marine rotifer B. plicatilis. In B. calyciflorus, a 24 h-no observed effect concentration (NOEC-24 h) and a 24 h-median lethal concentration (LC50-24 h) of chlordecone were determined as 100 μg/L and 193.8 μg/L, respectively, while NOEC-24 h and LC50-24 h of fipronil were determined as 1000 μg/L and 2033.0 μg/L, respectively. In B. plicatilis, NOEC-24 h and LC50-24 h of chlordecone were 100 μg/L and 291.0 μg/L, respectively, while NOEC-24 h and LC50-24 h of fipronil were determined as 1000 μg/L and 5735.0 μg/L, respectively. Moreover, retardation in the population growth were observed in response to chlordecone and fipronil in both rotifer species, suggesting that chlordecone and fipronil have a potential adverse effects on life cycle parameters of two rotifer species. Additionally, modulation in the expressions of the entire CYP genes were demonstrated in response to chlordecone and fipronil at 24 h period. These results provide the better understanding on how chlordecone and fipronil can affect in population growth of two rotifers and CYP gene expressions in chlordecone- and fipronil-exposed rotifers.
Species-specific behaviours in amphipods highlight the need for understanding baseline behaviours in ecotoxicology Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-19 Shanelle A. Kohler, Matthew O. Parker, Alex T. Ford
Behavioural studies in ecotoxicology are increasing with techniques and endpoints used in pharmacology being translated to other vertebrate and invertebrate species. Despite this, data on the baseline behaviours of model organisms, and inter-species variability in behaviour are currently under-studied. This study assessed a range of behaviours associated with anxiety including swimming speed, phototaxis and thigmotaxis in a marine and freshwater amphipod (Echinogammarus marinus and Gammarus pulex). Differences in sensitivity to these assays were observed between species with E. marinus showing a greater sensitivity to the phototaxis assay than G. pulex, while in thigmotaxis assays G. pulex appeared better suited than E. marinus for measuring differences in the use of central zones. Significant inter-species differences were also observed in swimming patterns when breaking the data into ten second time bins but not when data was broken into two-minute time bins. The results of this study provide evidence of phototactic and thigmotactic behaviours in two model crustacean species with potential for use in behavioural ecotoxicology. Inter-species variability in sensitivity to behavioural assays highlights the importance of systematic assessment of baseline responses for all model species used in behavioural studies. Careful analysis of data is also required when performing behavioural studies so as not to lose sensitivity in your data.
Toxicogenomic responses of low level anticancer drug exposures in Daphnia magna Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-19 Chiara Russo, Marina Isidori, Jessica A. Deaver, Helen C. Poynton
The use of anticancer drugs in chemotherapy is increasing, leading to growing environmental concentrations of imatinib mesylate (IMA), cisplatinum (CDDP), and etoposide (ETP) in aquatic systems. Previous studies have shown that these anticancer drugs cause DNA damage in the crustacean Daphnia magna at low, environmentally relevant concentrations. To explore the mechanism of action of these compounds and the downstream effects of DNA damage on D. magna growth and development at a sensitive life stage, we exposed neonates to low level concentrations equivalent to those that elicit DNA damage (IMA: 2000 ng/L, ETP: 300 ng/L, CDDP: 10 ng/L) and performed transcriptomic analysis using an RNA-seq approach. RNA sequencing generated 14 million reads per sample, which were aligned to the D. magna genome and assembled, producing approximately 23,000 transcripts per sample. Over 90% of the transcripts showed homology to proteins in GenBank, revealing a high quality transcriptome assembly, although functional annotation was much lower. RT-qPCR was used to identify robust biomarkers and confirmed the downregulation of an angiotensin converting enzyme-like gene (ance) involved in neuropeptide regulation across all three anticancer drugs and the down-regulation of DNA topoisomerase II by ETP. RNA-seq analysis also allowed for an in depth exploration of the differential splicing of transcripts revealing that regulation of different gene isoforms predicts potential impacts on translation and protein expression, providing a more meaningful assessment of transcriptomic data. Enrichment analysis and investigation of affected biological processes suggested that the DNA damage caused by ETP and IMA influences cell cycle regulation and GPCR signaling. This dysregulation is likely responsible for effects to neurological system processes and development, and overall growth and development. Our transcriptomic approach provided insight into the mechanisms that respond to DNA damage caused by anticancer drug exposure and generated novel hypotheses on how these chemicals may impact the growth and survival of this ecologically important zooplankton species.
Silicon limitation reduced the adsorption of cadmium in marine diatoms Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-17 Jie Ma, Beibei Zhou, Dandan Duan, Yang Wei, Ke Pan
The nitrogen (N) and phosphorus (P) loadings in coastal waters have greatly increased due to anthropogenic input. However, the silicate levels have remained stable in the past decades, leading to nutrient status alternating from N limitation to Si limitation. Few studies have examined the effects of such nutrient imbalance on Cd accumulation in marine diatoms. In the present study, we provide multiple lines of evidence that Si limitation reduced the Cd binding capability in the marine diatom Nitzschia closterium. Diatom cells adapted to lower Si exposure had weaker mechanical strength and less negatively charged surfaces. Close examination of the cell surface revealed that Si shortage changed the biochemical composition of the cell surface and decreased the diatom’s silicification ability. The lower density of the silanol groups in the cell wall is the most likely reason for the weaker Cd adsorption ability observed in the Si limited diatom cells. This study demonstrates the significance of Si in controlling the metal accumulation in marine diatoms.
Waterborne exposure to low concentrations of BDE-47 impedes early vascular development in zebrafish embryos/larvae Aquat. Toxicol. (IF 3.884) Pub Date : 2018-07-17 Xiumei Xing, Jianmeng Kang, Jiahuang Qiu, Xiali Zhong, Xiongjie Shi, Bingsheng Zhou, Yanhong Wei
Polybrominated diphenyl ethers (PBDEs) are persistent flame retardants ubiquitously existing in various environment matrices. In spite of a recent reduction in use according to the phase-out policy, high levels of PBDEs are still found in both environmental and biological samples due to their persistent property and large-scale production over a long history. Developmental toxicity is a major health concern of PBDEs. However, the impact of PBDE exposure on vascular development remains poorly understood. In this study, we investigated the effect of low concentrations of 2,2’,4,4’-Tetrabromodiphenyl ether (BDE-47), a predominant PBDE congener, in environmental matrices and biota, on early vascular development using zebrafish. Zebrafish embryos were continuously exposed to waterborne BDE-47 at 0.06, 0.2, 0.6 μM starting from 2 hours post-fertilization (hpf). Fluorescent images of vasculatures in Tg(kdrl:eGFP) zebrafish were acquired using a confocal microscope. The results indicated that BDE-47 exposure had no effect on hatching rate, survival, body weight, body length or heart rate in the early stage within 72 hpf, whereas zebrafish exposed to BDE-47 exhibited impairments in the growth of multiple types of blood vessels. The percentage of completed intersegmental vessels (ISV) at 30 hpf decreased in embryos treated with BDE-47 in a dose-dependent fashion. BDE-47 exposure led to a slight decrease in the growth of common cardinal vein (CCV), while dramatically hindered CCV remodeling process reflected by the larger CCV area and wider ventral diameter. BDE-47 exposure significantly reduced sub-intestinal vessels (SIV) area as well as the vascularized yolk area in zebrafish larvae at 72 hpf. In addition, the expression of genes related to vascular growth and remodeling was markedly suppressed in BDE-47-exposed zebrafish. These findings demonstrate the adverse effects of BDE-47 on early vascular development, and confirm the vascular toxicity of PBDEs in vivo. The results indicate that developing vasculature in zebrafish is sensitive to BDE-47 exposure, and may serve as a powerful tool for the assessment of early exposure to PBDEs.
Transcriptional effects of androstenedione and 17α-hydroxyprogesterone in zebrafish embryos Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-22 Karl Fent, Patricia Franziska Siegenthaler, Andreas Alexandre Schmid
Steroid hormones in the aquatic environment may pose a risk to fish health. Here we evaluated effects of two different class steroids that frequently occur in the aquatic environment, the androgen androstenedione (A4) and the progestin 17α-hydroxyprogesterone (17-OHP4). Zebrafish embryos were exposed to four concentrations of A4 and the positive control testosterone and to 17-OHP4, and transcriptional changes were determined at 96 h post fertilization (hpf) and 120 hpf. Transcriptional changes of 18 selected genes were assessed upon exposure to measured concentrations of 0.004, 0.046, 0.62 and 6.56 μg/L A4. Significant induction of the genes encoding sulfotransferase (sult2st3) and aromatase (cyp19b) occurred in 120 hpf embryos at 6.56 μg/L A4 and 1 μg/L testosterone. Additionally, cyp2k7 was significantly induced in two of three independent experiments. 17-OHP4 did not induce physiological effects (muscle contraction, heart rate, hatching success, swimming activity) at concentrations between 0.01 and 10 μg/L. Of the analyzed 15 genes, slight transcriptional alterations occurred for the genes encoding progesterone receptor, aromatases (cyp19a) and (cyp19b) and cyp2k7 at 10 μg/L. Our study highlights sult2st3, cyp19b and cyp2k7 as potential markers of androgen exposure in fish and indicates that 17-OHP4 is not likely to pose a risk for fish at environmental concentrations.
Can the inhibition of cytochrome P450 in aquatic invertebrates due to azole fungicides be estimated with in silico and in vitro models and extrapolated between species? Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-26 Michele Gottardi, Jonathan D. Tyzack, Andreas Bender, Nina Cedergreen
Azole fungicides, designed to halt fungal growth by specific inhibition of fungal cytochrome P450 (CYP51), inhibit cytochrome P450s involved in the metabolism of xenobiotics in several non-target organisms thus raising environmental concern. The present study investigates the degree by which inhibition strengths of azoles toward cytochrome P450 in rat liver, the insect Chironomus riparius larvae and the snail Lymnaea stagnalis can be extrapolated from estimated in silico affinities. Azoles’ affinities toward human cytochrome P450 isoforms involved in xenobiotic metabolism (CYP3A4, CYP2C9 and CYP2D6) as well as fungal CYP51 were estimated with a ligand-protein docking model based on the ChemScore scoring function. Estimated affinities toward the selected enzymatic structures correlated strongly with measured inhibition strengths in rat liver (ChemScore vs. logIC50 among cytochrome P450 isoforms: −0.662 < r < −0.891, n = 17 azoles), while weaker correlations were found for C. riparius larvae (−0.167 < r < −0.733, n = 9) and L. stagnalis (−0.084 < r < −0.648, n = 8). Inhibition strengths toward C. riparius and rat liver activities were found to be highly correlated to each other (r: 0.857) while no significant relationship was found between either of the species and L. stagnalis. The inhibition of cytochrome P450 due to azole fungicides could be estimated in vitro and to a lesser extent in silico for C. riparius but not for L. stagnalis, possibly due to different enzymatic susceptibility toward azole inhibition among the species.
Copper oxide nanoparticles induce the transcriptional modulation of oxidative stress-related genes in Arbacia lixula embryos Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-14 Alessia Giannetto, Tiziana Cappello, Sabrina Oliva, Vincenzo Parrino, Giuseppe De Marco, Salvatore Fasulo, Angela Mauceri, Maria Maisano
Copper oxide nanoparticles (CuO NPs) are widely used in various industrial applications, i.e. semiconductor devices, batteries, solar energy converter, gas sensor, microelectronics, heat transfer fluids, and have been recently recognized as emerging pollutants of increasing concern for human and marine environmental health. Therefore, the toxicity of CuO NPs needs to be thoroughly understood. In this study, we evaluated the potential role of oxidative stress in CuO NP toxicity by exploring the molecular response of Arbacia lixula embryos to three CuO NP concentrations (0.7, 10, 20 ppb) by investigating the transcriptional patterns of oxidative stress-related genes (catalase and superoxide dismutase) and metallothionein, here cloned and characterized for the first time. Time- and concentration-dependent changes in gene expression were detected in A. lixula embryos exposed to CuO NPs, up to pluteus stage (72 h post-fertilization, hpf), indicating that oxidative stress is one of the toxicity mechanisms for CuO NPs. These findings provide new insights into the comprehension of the molecular mechanisms underlying copper nanoparticle toxicity in A. lixula sea urchin and give new tools for monitoring of aquatic areas, thus corroborating the suitability of this embryotoxicity assay for future evaluation of impacted sites.
RNA-Seq analysis of transcriptome responses in Atlantic cod (Gadus morhua) precision-cut liver slices exposed to benzo[a]pyrene and 17α-ethynylestradiol Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-07 Fekadu Yadetie, Xiaokang Zhang, Eileen Marie Hanna, Libe Aranguren-Abadía, Marta Eide, Nello Blaser, Morten Brun, Inge Jonassen, Anders Goksøyr, Odd André Karlsen
Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) that activate the aryl hydrocarbon receptor (Ahr) pathway, and endocrine disruptors acting through the estrogen receptor pathway are among environmental pollutants of major concern. In this work, we exposed Atlantic cod (Gadus morhua) precision-cut liver slices (PCLS) to BaP (10 nM and 1000 nM), ethynylestradiol (EE2) (10 nM and 1000 nM), and equimolar mixtures of BaP and EE2 (10 nM and 1000 nM) for 48 h, and performed RNA-Seq based transcriptome mapping followed by systematic bioinformatics analyses. Our gene expression analysis showed that several genes were differentially expressed in response to BaP and EE2 treatments in PCLS. Strong up-regulation of genes coding for the cytochrome P450 1a (Cyp1a) enzyme and the Ahr repressor (Ahrrb) was observed in BaP treated PCLS. EE2 treatment of liver slices strongly up-regulated genes coding for precursors of vitellogenin (Vtg) and eggshell zona pellucida (Zp) proteins. As expected, pathway enrichment and network analysis showed that the Ahr and estrogen receptor pathways are among the top affected by BaP and EE2 treatments, respectively. Interestingly, two genes coding for fibroblast growth factor 3 (Fgf3) and fibroblast growth factor 4 (Fgf4) were up-regulated by EE2 in this study. To our knowledge, the fgf3 and fgf4 genes have not previously been described in relation to estrogen signaling in fish liver, and these results suggest the modulation of the FGF signaling pathway by estrogens in fish. The signature expression profiles of top differentially expressed genes in response to the single compound (BaP or EE2) treatment were generally maintained in the expression responses to the equimolar binary mixtures. However, in the mixture-treated groups, BaP appeared to have anti-estrogenic effects as observed by lower number of differentially expressed putative EE2 responsive genes. Our in-depth quantitative analysis of changes in liver transcriptome in response to BaP and EE2, using PCLS tissue culture provides further mechanistic insights into effects of the compounds. Moreover, the analyses demonstrate the usefulness of PCLS in cod for omics experiments.
Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-08 Reyna Cristina Colli-Dula, Xiefan Fang, David Moraga-Amador, Nacira Albornoz-Abud, Roberto Zamora-Bustillos, Ana Conesa, Omar Zapata-Perez, Diego Moreno, Emanuel Hernandez-Nuñez
Despite a wide number of toxicological studies that describe benzo[a]pyrene (BaP) effects, the metabolic mechanisms that underlie these effects in fish are largely unknown. Of great concern is the presence of BaP in aquatic systems, especially those in close proximity to human activity leading to consumption of potentially contaminated foods. BaP is a known carcinogen and it has been reported to have adverse effects on the survival, development and reproduction of fish. The purpose of this study was to investigate if a low dose of BaP can alter genes and key metabolic pathways in the liver and testis in male adult tilapia, and whether these could be associated with biological endpoints disruption. We used both high-throughput RNA-Sequencing to assess whole genome gene expression following repeated intraperitoneal injections of 3 mg/kg of BaP (every 6 days for 26 days) and morphometric endpoints as indicators of general health. Condition factor (K) along with hepatosomatic and gonadosomatic indices (morphometric parameters) were significantly lower in BaP-treated fish than in controls. BaP exposure induced important changes in the gene expression pattern in liver and testis as revealed by both Pathway and Gene Ontology (GO) analyses. Alterations that were shared by both tissues included arachidonic acid metabolism, androgen receptor to prostate-specific antigen signaling, and insulin-associated effects on lipogenesis. The most salient liver-specific effects included: biological processes involved in detoxification, IL6-associated insulin resistance, mTOR hyperactivation, mitotic cytokinesis, spindle pole and microtubule binding. BaP effects that were confined to the testis included: immune system functions, inflammatory response, estrogen and androgen metabolic pathways. Taken together, gene expression and morphometric end point data indicate that the reproductive success of adult male tilapia could be compromised as a result of BaP exposure. These results constitute new insights on the mechanism of action of low dose BaP in a non-model organism (tilapia).
New toxicogenetic insights and ranking of the selected pharmaceuticals belong to the three different classes: A toxicity estimation to confirmation approach Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-09 Yi Liu, Muhammad Junaid, Yan Wang, Yu-Mei Tang, Wan-Ping Bian, Wen-Xu Xiong, Hai-Yang Huang, Chun-Di Chen, De-Sheng Pei
Transcriptomics provides mechanistic indicators of fluoride toxicology on endochondral ossification in the hind limb of Bufo gargarizans Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-10 Wu Chao, Yuhui Zhang, Lihong Chai, Hongyuan Wang
Endochondral ossification, the process by which most of the bone is formed, is regulated by many specific groups of molecules and extracellular matrix components. Hind limb of Bufo gargarizans is a model to study endochondral ossification during metamorphosis. Chinese toad (Bufo gargarizans) were exposed to different fluoride concentrations (0, 1, 5, 10 and 20 mg L−1) from G3 to G42. The development of hind limb of B. gargarizans was observed using the double staining methodology. The transcriptome of hind limb of B. gargarizans was conducted using RNA-seq approach, and differentially expressed gene was also validated. In addition, the location of Sox9 and Ihh in the growth cartilage was determined using in situ hybridization. Our results showed that 5 mg L−1 stimulated bone mineralization, while 10 and 20 mg L−1 exposure could inhibit the tibio-fibula, tarsus and metacarpals ossification. Besides, 10 mg F/L treatment could down-regulate Ihh, Sox9, D2, D3, TRα, TRβ, Wnt10, FGF3 and BMP6 expression, while up-regulate ObRb and HHAT mRNA expression in the hind limb of B. gargarizans. Transcript level changes of Ihh, Sox9, D2, D3, TRα, TRβ, Wnt10, FGF3 and BMP6 were consistent with the results of RT-qPCR. In situ hybridization revealed that Ihh was expressed in prehypertrophic chondrocytes, while Sox9 was abundantly expressed in proliferous, prehypertrophic and hypertrophic chondrocytes. However, 10 mg F-/L did not cause any affect in the location of the Ihh and Sox9 mRNA. Therefore, high concentration of fluoride could affect the ossification-related genes mRNA expression and then inhibit the endochondral ossification. The present study thus will greatly contribute to our understanding of the effect of environmental contaminant on ossification in amphibian.
Influence of pH on the uptake and toxicity of β-blockers in embryos of zebrafish, Danio rerio Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-28 Lisa Bittner, Elisabet Teixido, Bettina Seiwert, Beate I. Escher, Nils Klüver
ß-Blockers are weak bases with acidity constants related to their secondary amine group. At environmental pH they are protonated with the tendency to shift to their neutral species at more alkaline pH. Here we studied the influence of pH from 5.5 to 8.6 on the toxicity of the four ß-blockers atenolol, metoprolol, labetalol and propranolol in zebrafish embryos, relating toxicity not only in a conventional way to external aqueous concentrations but also to measured internal concentrations. Besides lethality, we evaluated changes in swimming activity and heartbeat, using the Locomotor Response (LMR) method and the Vertebrate Automated Screening Technology (VAST) for high throughput imaging. Effects of metoprolol, labetalol and propranolol were detected on phenotype, heart rate and swimming activity. External effect concentrations decreased with increasing neutral fraction for all three pharmaceuticals, attributed by an enhanced uptake of the neutral species in comparison to the corresponding charged form. The LC50 of metoprolol decreased by a factor of 35 from 1.91 mM with almost complete cationic state at pH 7.0 to 0.054 mM with 8% neutral fraction at pH 8.6. For propranolol the LC50 of 2.42 mM at pH 5.5 was even 100 fold higher than the LC50 at pH 8 with 0.023 mM where 3% were neutral fraction. No effects were detected in the zebrafish embryo exposed to atenolol. The internal concentrations for metoprolol and propranolol were quantified at non-toxic concentrations and at the LC10. Apparent bioconcentration factors (BCF) ranged from 1.96 at pH 7.0 to 32.0 at pH 8.6 for metoprolol and from 1.86 at pH 5.5 to 169 at pH 8.0 for propranolol. The BCFs served to predict the internal effect concentrations from the measured external effect concentrations. Internal effect concentrations of metoprolol and propranolol were in a similar range for all pH-values and for all endpoints. Interestingly, the internal effect concentrations were in the internal concentration range of baseline toxicity, which suggests that the effects of the ß-blockers are rather unspecific, even for sublethal effects on heart rate. In summary, our data confirm that the pH-dependent toxicity related to external concentrations can be explained by toxicokinetic effects and that the internal effect concentrations are pH-independent.
Photo-induced antibacterial activity of a porphyrin derivative isolated from the harmful dinoflagellate Heterocapsa circularisquama Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-07 Li Wencheng, Kichul Cho, Yasuhiro Yamasaki, Satoshi Takeshita, Kiju Hwang, Daekyung Kim, Tatsuya Oda
The dinoflagellate Heterocapsa circularisquama is highly toxic to bivalves. However, significant toxicity to finfish species has not been reported. We previously found that H. circularisquama has light-dependent haemolytic agents. Purification and chemical structural analyses revealed that the haemolytic agent H2-a is a porphyrin derivative, which exhibits light-dependent cytotoxicity toward tumour cells. To clarify the biological activity of H2-a further, its antibacterial activities against Gram-positive and Gram-negative bacteria were investigated in this study. A fraction (F5) equivalent to H2-a purified from the methanol extract of H. circularisquama showed potent light-dependent bactericidal activity toward Staphylococcus aureus, and the activity was concentration- and light illumination time-dependent; however, Escherichia coli was highly resistant to F5. Electron microscopic observation suggested that F5 induces morphological changes in S. aureus in a light-dependent manner. Further analysis using other bacterial species showed that the Gram-positive bacterium Bacillus subtilis was more sensitive than the Gram-negative bacteria Pseudomonas aeruginosa and Vibrio alginolyticus. These results indicate that F5 is a photo-induced antibacterial agent with relatively higher specificity to Gram-positive bacteria. Iodometric assay suggested that singlet oxygen was generated from light-illuminated F5. Histidine, a specific singlet oxygen scavenger, markedly inhibited the photosensitising antibacterial activity of F5 against S. aureus, suggesting the involvement of singlet oxygen in antibacterial activity. The antibacterial spectrum of F5 was evidently different from that of 5,10,15,20-tetra (N,N,N-trimethylanilinium) porphyrin tetratosylate, a commercially available porphyrin compound with antibacterial activity. Our results demonstrate that H. circularisquama has a novel antibacterial photosensitiser, a porphyrin derivative, with relatively higher specificity to Gram-positive bacteria. To the best of our knowledge, this is the first study to discover a porphyrin derivative with antibacterial activity in marine microalga.
Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-06 Gabrielle do Amaral e Silva Müller, Karim Hahn Lüchmann, Guilherme Razzera, Guilherme Toledo-Silva, Maria João Bebianno, Maria Risoleta Freire Marques, Afonso Celso Dias Bainy
Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two–dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30–75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, β-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.
Immune response induced by major environmental pollutants through altering neutrophils in zebrafish larvae Aquat. Toxicol. (IF 3.884) Pub Date : 2018-06-06 Hongyan Xu, Xiaoyan Zhang, Hankun Li, Caixia Li, Xiao-Jing Huo, Li-Ping Hou, Zhiyuan Gong
Environmental pollutants may cause adverse effects on the immune system of aquatic organisms. However, the cellular effects of pollutants on fish immune system are largely unknown. Here, we exploited the transgenic zebrafish Tg(lysC:DsRed2) larva as a preliminary screening system to evaluate the potential inflammatory effects of environmental pollutants. Tg(lysC:DsRED2) larvae aged 7-day-postfertilization (7 dpf) were treated with selected environmental chemicals for 24 h (24 h) and the number of neutrophils were quantified using both image analysis and fluorescence activated cell sorting (FACS). We found that the numbers of neutrophils in the Tg(lysC:DsRED2) larvae were significantly increased by most of the organic chemicals tested, including E2 (17β-estradiol), BPA (Bisphenol-A), NDEA (N-nitrosodiethylamine), 4-NP (4-Nitrophenol) and Lindane (γ-hexachlorocyclohexane). Neutrophil numbers were also increased by all the metals tested (Na2HAsO4· 7H2O, Pb(NO3)2, HgCl2, CdCl2, CuSO4·5H2O, ZnSO4, and K2Cr2O7). The only exception was TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), which significantly reduced the number of neutrophils after exposure. Additionally, the transcription of genes (lyz, mpo, tnfα and il8) related to fish immune system were significantly modulated upon exposure to some of the selected chemicals such as E2, TCDD, Cu and Cd. This study revealed that representatives of major categories of environmental pollutants could cause an acute inflammatory response in zebrafish larvae as shown by alterations in the neutrophils, which may imply a common immunotoxicity mechanism for most environmental pollutants. This study has also demonstrated that Tg(lyz:DsRed2) transgenic zebrafish is an excellent tool for screening environmental chemicals with potential inflammatory effects through FACS-facilitated neutrophil counting.
Phosphate alleviation of glyphosate-induced toxicity in Hydrocharis dubia (Bl.) Backer Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-30 Guidi Zhong, Zhonghua Wu, Nian Liu, Jun Yin
Glyphosate, as a broad-spectrum herbicide, is frequently detected in water, and phosphorus widely enters the water due to the extensive use of phosphorus-containing substances in agriculture, industries and daily life. Thus, aquatic ecosystems are exposed to both glyphosate and phosphorus, which may affect aquatic organisms. In the present research, we studied the physiological responses of the floating aquatic plant species H. dubia to different concentrations of glyphosate (0, 1, 5, 15 mg/L) with different levels of phosphate (0, 50, 100 mg/L) after 14 days (d) of treatment. We explored glyphosate toxicity in H. dubia and investigated whether phosphate addition mitigates glyphosate toxicity in this species, which will provide a theoretical basis for the ecotoxicological study of aquatic plants. The results show that glyphosate significantly reduced the chlorophyll content, leaf number and root length of H. dubia, while it significantly increased the malondialdehyde (MDA), hydrogen peroxide (H2O2), shikimate, proline, and soluble protein content and enzyme activities (superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and polyphenol oxidase (PPO)) in H. dubia. After phosphate supplement, the MDA, H2O2, proline, and soluble protein contents and enzyme activities in the plants treated with glyphosate decreased. These results indicate that the concentration of glyphosate investigated in our study can cause oxidative stress and affect the growth of H. dubia. Phosphate can alleviate glyphosate-induced oxidative stress in H. dubia.
Comparative effects of cadmium, zinc, arsenic and chromium on olfactory-mediated neurobehavior and gene expression in larval zebrafish (Danio rerio) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-28 Kevin Heffern, Keith Tierney, Evan P. Gallagher
Studies have shown that olfactory-mediated behaviors that are critical to survival can be disrupted by exposure to certain metals. Polluted waterways often contain elevated levels of metals, yet only a subset have been characterized for their potential to cause olfactory toxicity. A larval zebrafish behavioral assay was developed to characterize concentration-response curves for zinc (Zn), hexavalent chromium (Cr), and arsenate (As) olfaction inhibition. Cadmium (Cd), an established olfactory toxicant, was used as a positive control. As expected, following a 24-hour exposure to Cd, we observed a reduced response to taurocholic acid (TCA), a substrate for ciliated olfactory sensory neurons (OSNs), thus validating the behavioral assay. Zn exposure similarly decreased the olfactory response toward TCA, (IC50: 36 μg/L and 76 μg/L, for Cd and Zn, respectively). The response towards a secondary odorant L-cysteine (Cys), a substrate for ciliated and microvillous OSNs, was significantly altered by both Cd and Zn exposure, although the response to Cys was not completely removed in Zn treated larvae, suggesting preferential toxicity towards ciliated OSNs. No significant changes in olfactory responses were observed following Cr and As exposures. Exposures to binary mixtures of Cd and Zn indicated that Zn had a protective effect against Cd toxicity at low Zn concentrations. QuantiGene (QDP) RNA analysis revealed Cd to be a potent inducer of metallothionein 2 (mt2) mRNA in zebrafish larvae, and Zn to be a weak mt2 inducer, suggesting a protective role of mt2 in Cd and Zn olfactory injury. By contrast, QDP analysis of eight other genes important in mitigating the effects of oxidative stress suggested an antioxidant response to Cd, but not Zn, As, and Cr suggesting that oxidative stress was not a primary mechanism of Zn-induced olfactory dysfunction. In summary, our study indicates that Zn inhibits zebrafish olfaction at environmental concentrations and may potentially mitigate Cd induced olfactory dysfunction when present in mixtures. The zebrafish behavioral trough assay incorporating the odorants L-cysteine and TCA is an effective assay to assess the effects of metals on olfactory function.
Genome-wide identification of 99 autophagy-related (Atg) genes in the monogonont rotifer Brachionus spp. and transcriptional modulation in response to cadmium Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-30 Hye-Min Kang, Jin-Sol Lee, Min-Sub Kim, Young Hwan Lee, Jee-Hyun Jung, Atsushi Hagiwara, Bingsheng Zhou, Jae-Seong Lee, Chang-Bum Jeong
Autophagy originated from the common ancestor of all life forms, and its function is highly conserved from yeast to humans. Autophagy plays a key role in various fundamental biological processes including defense, and has developed through serial interactions of multiple gene sets referred to as autophagy-related (Atg) genes. Despite their significance in metazoan life and evolution, few studies have been conducted to identify these genes in aquatic invertebrates. In this study, we identified whole Atg genes in four Brachionus rotifer spp., namely B. calyciflorus, B. koreanus, B. plicatilis, and B. rotundiformis, through searches of their entire genomes; and we annotated them according to the yeast nomenclature. Twenty-four genes orthologous to yeast genes were present in all of the Brachionus spp. while three additional gene duplicates were identified in the genome of B. koreanus, indicating that these genes had diversified during the speciation. Also, their transcriptional responses to cadmium exposure indicated regulation by cadmium-induced oxidative-stress–related signaling pathways. This study provides valuable information on 99 conserved Atg genes involved in autophagosome formation in Brachionus spp., with transcriptional modulation in response to cadmium, in the context of the role of autophagy in the damage response.
Comparative toxicity of three phenolic compounds on the embryo of fathead minnow, Pimephales promelas Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-29 Erchao Li, Derek G. Bolser, Kevin J. Kroll, Erica K. Brockmeier, Francesco Falciani, Nancy D. Denslow
Phenols are classified as polar narcotics, which are thought to cause toxicity by non-specific mechanisms, possibly by disrupting membrane structure and function. Here we test three phenolic chemicals, phenol, 2,4-dichlorphenol and pentachlorophenol on embryo development, heartbeat rate and mitochondrial respiration in fathead minnow (Pimephales promelas). While these chemicals have been used on isolated mitochondria, they have not yet been used to verify respiration in intact embryos. Mitochondrial respiration in intact embryos was measured after optimizing the Seahorse XFe24 Extracellular Flux Analyzer. Heartbeat rate and mitochondrial respiration patterns of fathead minnow embryos at different developmental stages were also characterized. Exposures of embryos at developmental stage 20 occurred for 24 h with five concentrations of each phenolic compound ranging from 0.85 to 255 μM for phenol, 0.49 to 147 μM for 2,4-dichlorophenol and 0.3 to 90 μM for pentachlorophenol. Exposure to phenol at the concentrations tested had no effects on development, heartbeat or mitochondrial respiration. However, both 2,4-dichlorophenol and pentachlorophenol showed dose-dependent effects on development, heartbeat rate, and mitochondrial respiration, with the effects occurring at lower concentrations of pentachlorophenol, compared to 2,4-dichlorophenol, highlighting the higher toxicity of the more chlorinated phenols. Both 2,4-dichlorophenol and pentachlorophenol decreased basal mitochondrial respiration of embryos and ATP production. These results indicate that higher chlorinated phenolic chemicals cause developmental toxicity in fathead minnow embryos by decreasing mitochondrial respiration and heartbeat rate.
Styrene impairs normal embryo development in the Mediterranean mussel (Mytilus galloprovincialis) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-30 Rajapaksha Haddokara Gedara Rasika Wathsala, Silvia Franzellitti, Morena Scaglione, Elena Fabbri
This study analysed the effects of styrene, a main monomer in plastic manufacturing and acknowledged to be amongst the most common plastic leachates, on early embryo development of the Mediterranean mussel. Embryotoxicity tests showed that styrene impaired normal embryo development at concentrations (0.01 μg/L–1 mg/L) encompassing the environmental range. Occurrence of normal D-veligers was significantly reduced up to 40% of the total, and larval size was reduced of about 20%. D-veligers grown in the presence of styrene (0.1 and 10 μg/L) showed significant reduction of total Multixenobiotic resistance (MXR) efflux activity that was not apparently related to transcriptional expression of genes encoding P-glycoprotein (ABCB) and Mrp (ABCC), the two main ABC transporters of embryonal MXR system. Indeed, ABCB transcription was not affected by styrene, while ABCC was up-regulated. At these same concentrations, transcriptional profiles of 15 genes underlying key biological functions in embryo development and potential targets of adverse effects of styrene were analysed. Main transcriptional effects were observed for genes involved in shell biogenesis and lysosomal responses (down-regulation), and in neuroendocrine signaling and immune responses (up-regulation). On the whole, results indicate that styrene may affect mussel early development through dysregulation of gene transcription and suggest the possible conservation of styrene mode of action across bivalve life cycle and between bivalves and humans, as well as through unpredicted impacts on protective systems and on shell biogenesis.
Chronic environmentally relevant levels of simvastatin disrupt embryonic development, biochemical and molecular responses in zebrafish (Danio rerio) Aquat. Toxicol. (IF 3.884) Pub Date : 2018-05-25 Susana Barros, Rosa Montes, José Benito Quintana, Rosario Rodil, Ana André, Ana Capitão, Joana Soares, Miguel M. Santos, Teresa Neuparth
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