Cellular bioenergetics is regulated by PARP1 under resting conditions and during oxidative stress

PurposeThe goal of the current studies was to elucidate the role of the principal poly(ADP-ribose)polymerase isoform, PARP1 in the regulation of cellular energetics in endothelial cells under resting conditions and during oxidative stress.MethodsWe utilized bEnd.3 endothelial cells and A549 human transformed epithelial cells. PARP1 was inhibited either by pharmacological inhibitors or by siRNA silencing. The Seahorse XF24 Extracellular Flux Analyzer was used to measure indices of mitochondrial respiration (oxygen consumption rate) and of glycolysis (extracellular acidification rate). Cell viability, cellular and mitochondrial NAD⁺ levels and mitochondrial biogenesis were also measured.ResultsSilencing of PARP1 increased basal cellular parameters of oxidative phosphorylation, providing direct evidence that PARP1 is a regulator of mitochondrial function in resting cells. Pharmacological inhibitors of PARP1 and siRNA silencing of PARP1 protected against the development of mitochondrial dysfunction and elevated the respiratory reserve capacity in endothelial and epithelial cells exposed to oxidative stress. The observed effects were unrelated to an effect on mitochondrial biogenesis. Isolated mitochondria of A549 human transformed epithelial cells exhibited an improved resting bioenergetic status after stable lentiviral silencing of PARP1; these effects were associated with elevated resting mitochondrial NAD⁺ levels in PARP1 silenced cells.ConclusionsPARP1 is a regulator of basal cellular energetics in resting endothelial and epithelial cells. Furthermore, endothelial cells respond with a decrease in their mitochondrial reserve capacity during low-level oxidative stress, an effect, which is attenuated by PARP1 inhibition. While PARP1 is a regulator of oxidative phosphorylation in resting and oxidatively stressed cells, it only exerts a minor effect on glycolysis.     

Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes

Triclosan (TCS) is an antimicrobial used widely in hospitals and personal care products, at ~10 mm . Human skin efficiently absorbs TCS. Mast cells are ubiquitous key players both in physiological processes and in disease, including asthma, cancer and autism. We previously showed that non‐cytotoxic levels of TCS inhibit degranulation, the release of histamine and other mediators, from rat basophilic leukemia mast cells (RBL‐2H3), and in this study, we replicate this finding in human mast cells (HMC‐1.2). Our investigation into the molecular mechanisms underlying this effect led to the discovery that TCS disrupts adenosine triphosphate (ATP) production in RBL‐2H3 cells in glucose‐free, galactose‐containing media (95% confidence interval EC₅₀ = 7.5–9.7 µm ), without causing cytotoxicity. Using these same glucose‐free conditions, 15 µm TCS dampens RBL‐2H3 degranulation by 40%. The same ATP disruption was found with human HMC‐1.2 cells (EC₅₀ 4.2–13.7 µm ), NIH‐3 T3 mouse fibroblasts (EC₅₀ 4.8–7.4 µm ) and primary human keratinocytes (EC₅₀ 3.0–4.1 µm ) all with no cytotoxicity. TCS increases oxygen consumption rate in RBL‐2H3 cells. Known mitochondrial uncouplers (e.g., carbonyl cyanide 3‐chlorophenylhydrazone) previously were found to inhibit mast cell function. TCS‐methyl, which has a methyl group in place of the TCS ionizable proton, affects neither degranulation nor ATP production at non‐cytotoxic doses. Thus, the effects of TCS on mast cell function are due to its proton ionophore structure. In addition, 5 µm TCS inhibits thapsigargin‐stimulated degranulation of RBL‐2H3 cells: further evidence that TCS disrupts mast cell signaling. Our data indicate that TCS is a mitochondrial uncoupler, and TCS may affect numerous cell types and functions via this mechanism. Copyright © 2015 John Wiley & Sons, Ltd.     

Directional and color preference in adult zebrafish: Implications in behavioral and learning assays in neurotoxicology studies

The zebrafish (Danio rerio) is a useful vertebrate model organism for neurological studies. While a number of behavior and learning assays are recently reported in the literature for zebrafish, many of these assays are still being refined. The initial purpose of this study was to apply a published T‐maze assay for adult zebrafish that measures how quickly an organism can discriminate between different color stimuli after receiving reinforcement to measure learning in a study investigating the later life impacts of developmental Pb exposure. The original results were inconclusive as the control group showed a directional and color preference. To assess directional preference further, a three‐chambered testing apparatus was constructed and rotated in several directions. The directional preference observed in males was alleviated by rotating the arms pointing west and east. In addition, color preference was investigated using all combinations of five different colors (orange, yellow, green, blue and purple). With directional preference alleviated results showed that both male and female zebrafish preferred colors of shorter wavelengths. An additional experiment tested changes in color preference due to developmental exposure to Pb in adult male zebrafish. Results revealed that Pb‐exposed males gained and lost certain color preferences compared to control males and the preference for short wavelengths was decreased. Overall, these results show that consideration and pretesting should be completed before applying behavioral and learning assays involving adult zebrafish to avoid innate preferences and confounding changes in neurotoxicology studies and that developmental Pb exposure alters color preferences in adult male zebrafish. Copyright © 2015 John Wiley & Sons, Ltd.     

Differential gene expression patterns during embryonic development of sea urchin exposed to triclosan

Triclosan (TCS; 2,4,4′‐trichloro‐2′‐hydroxydiphenyl ether) is a broad‐spectrum antibacterial agent used in common industrial, personal care and household products which are eventually rinsed down the drain and discharged with wastewater effluent. It is therefore commonly found in the aquatic environment, leading to the continual exposure of aquatic organisms to TCS and the accumulation of the antimicrobial and its harmful degradation products in their bodies. Toxic effects of TCS on reproductive and developmental progression of some aquatic organisms have been suggested but the underlying molecular mechanisms have not been defined. We investigated the expression patterns of genes involved in the early development of TCS‐treated sea urchin Strongylocentrotus nudus using cDNA microarrays. We observed that the predominant consequence of TCS treatment in this model system was the widespread repression of TCS‐modulated genes. In particular, empty spiracles homeobox 1 (EMX‐1), bone morphogenic protein, and chromosomal binding protein genes showed a significant decrease in expression in response to TCS. These results suggest that TCS can induce abnormal development of sea urchin embryos through the concomitant suppression of a number of genes that are necessary for embryonic differentiation in the blastula stage. Our data provide new insight into the crucial role of genes associated with embryonic development in response to TCS. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 426–433, 2017.     

Assessing the toxicity of the benzamide fungicide zoxamide in zebrafish (Danio rerio): Towards an adverse outcome pathway for beta-tubulin inhibitors

Commercial benzamide fungicides are applied to crops to control damage caused by oomycete fungi and are used as veterinary pharmaceuticals in aquaculture. The mechanism of action of these fungicides is to induce mitotic arrest via binding to beta-tubulin, thus inhibiting tubulin polymerization. However, there are little toxicity data available for benzimidazole fungicides in fish. To address this knowledge gap, we conducted zebrafish embryo toxicity tests to assess deformities, survival, and sub-lethal responses following exposure to zoxamide (0, 0.5, 1.0, 2.5, 5.0 and 10 μM zoxamide). We hypothesized that skeletal deformities would be prevalent in zebrafish due to its mechanism of inhibiting beta-tubulin polymerization. Zoxamide was relatively toxic to zebrafish embryos and larvae, and survival was reduced ∼50 % at 2 days post fertilization (dpf) with 10 μM exposure and over time at 6 dpf, 2.5 μM exposure reduced survival by ∼20 %. Frequency of hatch was also reduced/delayed in zebrafish at 3 dpf with >2.5 μM zoxamide. Pericardial edema, body length shortening, and spine curvature were observed in larvae exposed to >5 μM. Mitochondrial bioenergetics were assessed in ∼30 hpf embryos (24-hour exposure) using an XFe24 Flux Analyzer and regression analysis revealed a negative relationship between basal respiration and zoxamide concentration. Superoxide dismutase 1 and caspase 3 mRNA levels were both decreased in 6 dpf larvae exposed to 2.5 μM zoxamide, but were not changed in expression at 0.5 nor 1 μM zoxamide. Continuous 6-day exposure to zoxamide reduced larval activity at 2.5 μM; conversely a 24-hour exposure (at 5–6 dpf) induced hyperactivity at 5 μM suggesting dose and time dependent effects on fish behavior. Based on sub-lethal endpoints, we conceptualize an adverse outcome pathway for chemicals that inhibit tubulin polymerization.     

Early life stage and genetic toxicity of stannous chloride on zebrafish embryos and adults: Toxic effects of tin on zebrafish

Humans are exposed to stannous chloride (SnCl₂), known as tin chloride, present in packaged food, soft drinks, biocides, dentifrices, etc. Health effects in children exposed to tin and tin compounds have not been investigated yet. Therefore, we evaluated the possible teratogenic effects and genotoxic of SnCl₂ in zebrafish (Danio rerio) adults and their embryos. In the embryo–larval study, SnCl₂ showed embryo toxicity and developmental delay after exposure to the various concentrations of 10–250 μM for 120 h. Teratogenic effects including morphological malformations of the embryos and larvae were observed. The embryos exposed to 100 μM displayed tail deformation at 28 hpf and the larvae exposed to 50 μM showed reduced body growth, smaller head and eyes, bent trunk, mild pericardial edema, and smaller caudal fin at 96 hpf. The results of the teratological study show that SnCl₂ induced a significant decrease in the number of living embryos and larvae. Regarding the chromosome analysis, SnCl₂ induced a dose‐dependent increase in the micronucleus (MN) frequency in peripheral erythrocytes of adult zebrafish. In blood cells, the 25 μM dose of SnCl₂ caused a nonsignificant increase in the total chromosomal aberrations, but the high doses significantly increased the total number of chromosomal aberrations compared with the control groups. Overall, the results clearly indicate that SnCl₂ is teratogenic and genotoxic to zebrafish. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

Developmental toxicity assay using high content screening of zebrafish embryos

Typically, time‐consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post‐fertilization. Here we describe an automated image‐based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post‐acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth‐retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Developmental toxicity,EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

Dioxin‐like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L^?1 from 3‐h post‐fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non‐inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126‐induced developmental toxicity, we conducted ethoxyresorufin‐O‐deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real‐time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L^?1 concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L^?1 doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L^?1 at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose‐dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 201–210, 2016.     

Acute phase protein orosomucoid protects against heart failure through CCR5-mediated improvement in energy metabolism and calcium sensitivity

OBJECTIVE Improving energy metabolism is a promising target for the treatment of heart failure(HF). Our previous studies found that acute-phase protein ORM could increase glycogen content and muscle endurance in skeletal muscle via CCR5-AMPK pathway. Here,we further explored the effect and mechanism of ORM in HF. METHODS HF mice were induced by left anterior descending(LAD) coronary artery ligation(MI). ORM1 knockout mice were generated by our lab previously,ORM overexpression lentivirus(ov-ORM) was injected into the infarcted myocardium to overexpress ORM. Heart function was detected by echocardiography and hemodynamic parameters were measured by AD Instruments Power Lab data acquisition and analysis system. ORM expression was detected by Western blotting and ELISA.Cardiomyocytes were generated from human induced pluripotent stem cells(i PSC). iPSC cardiomyocyte contraction and viability were examined by Cardio Excyte-96. We used the whole cell voltage-clamp technique to record L-type Ca~(2+)currents. The specific binding of ORM to CCR5 was examined by confocal microscopy. Glycogen,ATP and ATPase activity were detected by corresponding assay kit. We used a Seahorse XF96 Extracellular Flux Analyzer to measure oxygen consumption rate of i PSCCMs. We performed Ion Optixto assess cytosolic Ca~(2+) transient and myofilament sensitivity to Ca~(2+). RESULTS ORM expression in serum, myocardial and liver tissues was significantly elevated in HF mice induced by left anterior descending(LAD) coronary artery ligation(MI).Exogenous and endogenous ORM could significantly improve cardiac function, while ORM knockout showed opposite effect and shortened survival rate. In human induced pluripotent stem cel-derived cardiomyocytes(i PSCCMs), ORM treatment could promote its contraction without affecting beat rate, cell index and intracellular calcium.ORM was found to specifically bind to CCR5 in iPSCCMs. Blocking CCR5 with antagonist Maraviroc abolished ORM's effect in cardiac function and energy metabolism. Further studies found ORM increased calcium sensitivity, which was also blocked by CCR5 antagonist.CONCLUSION ORM might act as a novel endogenous inotropic protein. It could improve cardiac energy metabolism and increase calcium sensitivity via CCR5, indicating a potential therapeutic target for HF.     

A novel screening method for transition metal‐based anticancer compounds using zebrafish embryo‐larval assay and inductively coupled plasma‐mass spectrometry analysis

As novel metallodrugs continue to emerge, they are evaluated using models, including zebrafish, that offer unique sublethal endpoints. Testing metal‐based anticancer compounds with high‐throughput zebrafish toxicological assays requires analytical methods with the sensitivity to detect these sublethal tissue doses in very small sample masses (e.g., egg mass 100 μg). A robust bioanalytical model, zebrafish embryos coupled with inductively coupled plasma‐mass spectrometry (ICPMS) for measurement of delivered dose, creates a very effective means for screening metal‐based chemotherapeutic agents. In this study, we used ICPMS quantitation with the zebrafish embryo assays to detect metal equivalents at multiple response endpoints for two compounds, the chemotherapeutic agent cisplatin and ruthenium (Ru)‐based prospective metallodrug, PMC79. We hypothesized that cisplatin and PMC79 have different mechanisms for inducing apoptosis and result in similar lesions but different potencies following water‐borne exposure. An ICPMS method was developed to detect the metal in waterborne solution and tissue (detection limit: 5 parts per trillion for Ru or platinum [Pt]). The Ru‐based compound was more potent (LC₅₀: 7.8 μm ) than cisplatin (LC₅₀: 158 μm ) and induced disparate lesions. Lethality from cisplatin exposure exhibited a threshold (values >15 mg/L) while no threshold was observed for delayed hatching (lowest observed adverse effect level 3.75 mg/L cisplatin; 8.7 Pt (ng)/organism). The Ru organometallic did not have a threshold for lethality. Cisplatin‐induced delayed hatching was investigated further by larval‐Pt distribution and preferentially distributed to the chorion. We propose that zebrafish embryo‐larval assays coupled with ICPMS serve as a powerful platform to evaluate relative potency and toxic effects of metallodrug candidates.     

Exposure time to caffeine affects heartbeat and cell damage‐related gene expression of zebrafish Danio rerio embryos at early developmental stages

Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100 µm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48 h post‐fertilization (hpf) to 96 hpf. At 60 and 72 hpf, hatching decreased significantly (P < 0.05); however, the overall hatching rate at 96 hpf was 94% in control and 93% in caffeine treatment with no significant difference (P > 0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96 hpf, respectively. Caffeine significantly increased heartbeat – 122 and 136 at 72 and 96 hpf, respectively. Quantitative RT‐PCR showed significant up‐regulation after caffeine exposure in HSP70 at 72 hpf; in Cyclin G1 at 24, 48 and 72 hpf; and in Bax at 48 and 72 hpf. Significant down‐regulation was found in Bcl2 at 48 and 72 hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72 hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial‐dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell‐damage related genes. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early retinal development in zebrafish, Danio rerio. Zebrafish embryos were immediately exposed to different concentrations (0, 0.125, 0.25, 0.5, 1.0 and 2.0 mg) of PCBs per liter of medium at 28.5 °C. Embryos were assessed at 30, 48, 72 and 96 h post‐fertilization (hpf) for changes in embryonic survival rate, development, larval retinal morphology and ultrastructure of the retina. The results show that PCB exposure decreased the survival rate of embryos in a time‐ and dose‐dependent manner. Embryos exposed to the higher concentrations of PCBs (0.5, 1.0 and 2.0 mg l^?1) displayed obvious gross morphological deformities. At 72 hpf, the retinal layer development of zebrafish was delayed at higher PCB concentrations (1.0 mg l^?1). At 96 hpf, irregularity of photoreceptor cells arrangement and thickening of photoreceptor and ganglionic layers were observed in PCB‐treated larvae at concentrations of 0.25–1 mg l^?1. Ultrastructural examination showed signs of growth inhibition of the photoreceptor outer segment at 0.25–1 mg l^?1 PCB exposure at 72 hpf, as well as the appearance of massive vacuoles and holes inside the outer segments in the PCB exposure group at 96 hpf. These results suggest that embryonic exposure to moderate and high levels of PCBs induced developmental deficits in zebrafish retinas, particularly in photoreceptor cells. Copyright © 2011 John Wiley & Sons, Ltd.     

PPARβ/δ-agonist F3SM and GW501516 ameliorates dysfunction in glycolysis in sAD patient iPSCs derived neural stem cells

OBJECTIVE To establish an in vitro cell model based on sAD patient-specific human neural stem cells and explore the energy metabolism of the neural stem cel s(NSCs), then observe whether PPARβ/δ-agonist F3SM and GW501516 can improve the energy metabolism of NSCs. METHODS The induced pluripotent stem cells(i PSCs) of sAD patients and healthy controls were first induced into neural stem cells. Then the differentiated NSCs were identified by immunofluorescence.The energy metabolism of NSCs was detected by Seahorse XF96 Extracellular Flux Analyzer(Seahorse Bioscience) and the rate of the extracellular pH drift(ECAR) was analyzed, which can reflect the glycolysis level of cells.RESULTS NSCs derived from sAD patients and healthy controls expressed neural stem cell markers Nestin,Sox1, Sox2 and Ki67. The analysis of ECAR showed that the basic glycolysis level and the maximum glycolysis capacity of the NSCs from healthy controls were significantly higher than those of sAD patients, which reflected abnormal energy metabolism of NSCs derived from sAD patients. While PPARβ/δ-agonist F3 SM and GW501516 can ameliorate dysfunction in glycolysisin sAD patient NSCs by enhancing the level of basic glycolysis and maximum glycolysis capacity. CONCLUSION On the basis of the experiments we conducted, we can conclude that sADi PSCs-derived NSCs exhibited dysfunction in energy metabolism, revealing the pathological characteristics of early sAD. PPARβ/δ-agonist F3SM and GW501516 can ameliorate dysfunctionin glycolysisin sAD patient NSCs,which might be served as new therapeutic strategy for AD.     

Proteomics analysis of zebrafish larvae exposed to 3,4‐dichloroaniline using the fish embryo acute toxicity test

The zebrafish (Danio rerio) is a small teleost fish that is becoming increasingly popular in laboratories worldwide and several attributes have also placed the zebrafish under the spotlight of (eco)toxicological studies. Since the 1990s, international organizations such as ISO and OECD have published guidelines for the use of zebrafish in ecotoxicological assessment of environmental toxicants such as the Fish Embryo Acute Toxicity (FET) test, OECD n° 236 guideline. This protocol uses 3,4‐dichloroaniline (DCA), an aniline pesticide whose toxicity to fish species at early life stages is well known, as a positive control. Despite its use, little is known about its molecular mechanisms, especially in the context of the FET test. Therefore, this study aimed to investigate such changes in zebrafish larvae exposed to DCA (4 mg/L) for 96 hours using gel‐free proteomics. Twenty‐four proteins detected in both groups were identified as significantly affected by DCA exposure, and, when considering group‐specific entities, 48 proteins were exclusive to DCA (group‐specific proteins) while 248 were only detected in the control group. Proteins modulated by DCA treatment were found to be involved in metabolic processes, especially lipids and hormone metabolism (eg, Apoa1 and Apoa1b and vitelogenins), as well as proteins important for developmental processes and organogenesis (eg, Myhc4, Acta2, Sncb, and Marcksb). The results presented here may therefore provide a better understanding of the relationships between molecular changes and phenotype in zebrafish larvae treated with DCA, the reference compound of the FET test.     

Zebrafish neurotoxicity from aphantoxins—cyanobacterial paralytic shellfish poisons (PSPs) from Aphanizomenon flos‐aquae DC‐1

Aphanizomenon flos‐aquae (A. flos‐aquae), a cyanobacterium frequently encountered in water blooms worldwide, is source of neurotoxins known as PSPs or aphantoxins that present a major threat to the environment and to human health. Although the molecular mechanism of PSP action is well known, many unresolved questions remain concerning its mechanisms of toxicity. Aphantoxins purified from a natural isolate of A. flos‐aquae DC‐1 were analyzed by high‐performance liquid chromatography (HPLC), the major component toxins were the gonyautoxins1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). The LD₅₀ of the aphantoxin preparation was determined to be 11.33 μg/kg (7.75 μg saxitoxin equivalents (STXeq) per kg) following intraperitoneal injection of zebrafish (Danio rerio). To address the neurotoxicology of the aphantoxin preparation, zebrafish were injected with low and high sublethal doses of A. flos‐aquae DC‐1 toxins 7.73 and 9.28 μg /kg (5.3 and 6.4 μg STXeq/kg, respectively) and brain tissues were analyzed by electron microscopy and RT‐PCR at different timepoints postinjection. Low‐dose aphantoxin exposure was associated with chromatin condensation, cell‐membrane blebbing, and the appearance of apoptotic bodies. High‐dose exposure was associated with cytoplasmic vacuolization, mitochondrial swelling, and expansion of the endoplasmic reticulum. At early timepoints (3 h) many cells exhibited characteristic features of both apoptosis and necrosis. At later timepoints apoptosis appeared to predominate in the low‐dose group, whereas necrosis predominated in the high‐dose group. RT‐PCR revealed that mRNA levels of the apoptosis‐related genes encoding p53, Bax, caspase‐3, and c‐Jun were upregulated after aphantoxin exposure, but there was no evidence of DNA laddering; apoptosis could take place by pathways independent of DNA fragmentation. These results demonstrate that aphantoxin exposure can cause cell death in zebrafish brain tissue, with low doses inducing apoptosis and higher doses inducing necrosis. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Investigation into the sub‐lethal effects of the triazole fungicide triticonazole in zebrafish (Danio rerio) embryos/larvae

Global use of azole fungicides is expected to increase over the next several years. Triticonazole is a triazole fungicide that is used for turf protection, residential, and other commercial applications. As such, it can enter local rural and urban water systems via run‐off and rain events. Early life stages of aquatic organisms can be susceptible to pesticides that enter the water, but in the case of triticonazole, data on the potential for subacute toxicity are lacking. Here, we determined the effects of triticonazole on development, oxygen consumption rates, and locomotor activity in zebrafish to address this knowledge gap. Wild‐type zebrafish (ABTu strain) embryos and larvae were exposed to triticonazole (1‐100 μM) in early development for different lengths of time depending on the assay conducted. Triticonazole did not affect survival nor induce significant deformity (pericardial edema, skeletal defects) in zebrafish at doses up to 100 μM. Oxygen consumption rate was measured in embryos after 24 and 48 hour exposure to triticonazole beginning at ～6 hpf using the XF_e flux analyzer. Triticonazole did not affect basal respiration, oligomycin‐induced ATP linked respiration, FCCP‐induced maximum respiration, proton leak, spare capacity, nor non‐mitochondrial respiration at doses up to 100 μM for 24 hours, even for exposure up to 250 μM for 48 hours. To determine whether the fungicide affected larval swimming activity, the visual motor response test was conducted following triticonazole exposure for 6 days. Larval zebrafish exposed to triticonazole showed hypoactivity in the dark following a 100 μM treatment, suggesting that the fungicide can affect the locomotor activity of zebrafish, albeit at relatively high levels. Given the fact that sublethal biological responses were absent at lower environmentally relevant concentrations, we conclude that triticonazole, relative to other triazole fungicides and types of pesticides, exhibits a relatively low risk of toxicity to the early life stages of fish.     

Methyl-triclosan and triclosan impact embryonic development of <Emphasis Type="Italic">Danio rerio</Emphasis> and <Emphasis Type="Italic">Paracentrotus lividus</Emphasis>

The presence of emerging pollutants in the environment is of major concern not only because of the potential negative impact in human health, but also due to the potential toxicity to non-target organisms. Within the personal and care products (PCPs), the disinfectant Triclosan (TCS) is one of the most concerning compounds. Once in the wastewater treatment plants (WWTPs), a small part of TCS can be biotransformed into a more persistent by-product: methyl-triclosan (M-TCS). Although several studies have focused on the occurrence of this compound in the water systems, the information on its toxicity to aquatic organisms is very limited. Here, we used embryo bioassays with two aquatic model animals to improve risk assessment of M-TCS; zebrafish (Danio rerio) embryo bioassays run up to 144?h post fertilization (hpf) and sea urchin (Paracentrotus lividus) up to 48?hpf, following established protocols. M-TCS and TCS exhibited similar toxicity to zebrafish with a NOEC of 160?µg/L. In contrast, M-TCS induced a delay in the development of the sea urchin larvae at all tested concentrations (1–1000?µg/L), whereas NOEC of TCS for P. lividus embryos was 40?µg/L. Overall, given the reported effects of M-TCS in the close range of environmentally relevant concentrations, and considering the low degradation rate and tendency to bioaccumulation (logKow: 5.2), further studies are warrant to better characterize the risk of this TCS metabolite to aquatic organisms.     

Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels

Trichlorfon (TCF) is one of the most used veterinary pharmaceuticals not only to fight infestations but also as a preventive measure worldwide. The high concentrations used generate concerns about environmental and human health. In this work we assessed the acute toxicity of this compound to non-target organisms belonging to different trophic levels: Danio rerio (early life stages and adults), Daphnia magna and algae (Pseudokirchneriella subcapitata and Chlorella vulgaris), and studied the potential of the biomarkers cholinesterase (ChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH) and catalase (CAT) to assess sub-lethal effects of trichlorfon in zebrafish and daphnids. The fish embryo test followed the OECD draft guideline FET and was based on the exposure of newly fertilized eggs to 0, 2.5, 5.0, 10, 20, 40, 80 and 160 mg/L of TCF for 5 days; the fish acute test followed the OECD guideline 203 and was based on the exposure of adult fish to 0, 2.5, 5, 10, 20, 40, 60 and 80 mg/L of TCF for 4 days; Daphnia sp. immobilization assay followed the OECD guideline 202 and was based on the exposure of juvenile daphnids to 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1 and 2 μg/L of TCF for 2 days and the algae growth inhibition assay followed the OECD guideline 201 and was based on the exposure of the two species to 0, 1, 3.2, 10, 32, 100 and 300 mg/L of TCF for 4 days. Biomarker levels were measured after 96 h exposure to TCF in zebrafish early life stages and adults and after 48 h exposure in D. magna. Tested organisms seem to have dissimilar sensitivities towards TCF exposure. D. magna (48 h-LC(50)=0.29 μg/L) was the most sensitive organism, followed by early life stages and adults of zebrafish (96 h-LC(50)=25.4 and 28.8 mg/L, respectively) and finally by the algae P. subcapitata (96 h-LC(50)=274.5 mg/L) and C. vulgaris (no effect observed). As daphnids are a source of food for organisms of higher trophic levels, the impairment on its population is prone to have consequences in the entire ecosystem. The biomarker activities measured in daphnids and fish seemed to be useful tools in the assessment of trichlorfon effects, especially ChE activity which was the most sensitive biomarker tested for all organisms. Trichlorfon was teratogenic for zebrafish embryos leading to anomalies in the absorption of the yolk sac, spine bending and pericardial oedemas. The present research suggests that further work is urgently needed in order to monitor environmental concentrations of trichlorfon and to test the long term effects of environmentally realistic concentrations of this compound.     

Toxicity effects of captan on different life stages of zebrafish (Danio rerio)

The objective of this study was to evaluate the toxicity and developmental effects of captan on different life stages (embryo and adult) of zebrafish (Danio rerio). The results showed that the 96-h lethal concentration 50 (LC₅₀) values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The results of developmental effect experiment showed that captan can significantly decrease the heartbeats and inhibit the hatching rate and growth of zebrafish embryos. Moreover, captan exposure can induce a series of deformities, including pericardial edema, yolk sac edema, spine curvature, and tail bending, in zebrafish embryos during the developmental period. Among these, the most significant were tail bending and spine curvature.     

Ecotoxicity and screening level ecotoxicological risk assessment of five antimicrobial agents: triclosan,triclocarban, resorcinol,phenoxyethanol and p‐thymol

Acute and chronic (or sub‐chronic) toxicity of five selected antimicrobial agents, including triclosan (TCS), triclocarban (TCC), resorcinol, phenoxyethanol and p‐thymol, was investigated using the conventional three‐aquatic‐organism battery. These compounds are widely used in cosmetics and other personal care products and their ecological risk has recently become a significant concern. As results of toxicity tests, TCS was found to be most strongly toxic for green algae [e.g. 72 h no observed effect concentration (NOEC) of 0.50 µg l^?1] among the selected compounds, followed by TCC, while TCC was more toxic or similar to TCS for Daphnia and fish (e.g. Daphnia 8 day NOEC of 1.9 µg l^?1). Having compared the predicted no effect concentration (PNEC) determined from the toxicity data with measured environmental concentrations (MEC), the preliminary ecological risk assessment of these five antimicrobials was conducted. The MEC/PNEC ratios of TCS and TCC were over 1 for some monitoring data, especially in urban streams with watershed areas without sewage service coverage, and their potential risk for green algae and Daphnia might be at a level of concern, although the contribution of TCS/TCC on the total toxicity of the those sites needs to be further investigated. For the three other antimicrobials, the maximum MEC/PNEC ratio for resorcinol was 0.1–1, but those for phenoxyethanol and p‐thymol were <0.1 and their risk to aquatic organisms is limited, although the additive effects with TCS, TCC and other antimicrobial agents, such as parabens, need to be further examined in future studies. Copyright © 2012 John Wiley & Sons, Ltd.