AOP-Anchored Transcriptome Analysis Catalogue Accelerates the Discovery of Environmental Toxicants in Zebrafish.

Current toxicity screening approaches to evaluate the vast number of environmental chemicals that require assessment are hampered due to their significant costs, time requirements, and reliance on live animal testing. The aim of the present study was to develop an adverse outcome pathway (AOP)-anchored transcriptome analysis (AATA) catalogue to expedite the discovery of environmental toxicants. 437 AOPs from the AOPwiki (https://aopwiki.org/) and 2280 transcriptomics data sets from NCBI Gene Expression Omnibus (GEO) and EMBL-EBI ArrayExpress (AE) repositories were comprehensively reviewed and analyzed. By using the differentially expressed molecular key event (mKE) genes as connection nodes, we created a large-scale environmental substance─target gene (mKE)─predicted adverse outcomes (SGAs) network that included 78 substances, 1099 genes, and 354 adverse outcomes (AOs). To validate the reliability of the network, comprehensive literature verification was conducted. We demonstrated that 164 of the 354 AOs identified have been previously characterized in the literature. The results for 136 of these AOs were consistent with the predictions of the AATA catalogue, representing an accuracy rate of 82.9%. Besides, distinct patterns in molecular KEs and AOs among categories of substances, such as biocides and metals, were demonstrated. Some representative substances, including atrazine and copper, pose significant risks to fish at various levels of biological organization. Moreover, experimental verification of the AATA predictions was conducted, including exposures of zebrafish to perfluorooctanesulfonate, cresyl diphenyl phosphate, and lanthanum. Results demonstrated consistency with predictions of the AATA catalogue, with an accuracy rate of 92.3%. Collectively, the present findings support the AATA catalogue as an efficient and promising platform for identifying environmental toxicants to fish and thereby provide novel insights into the understanding of potential risks of environmental contaminants.

Reduced Transcriptome Analysis of Zebrafish Embryos Prioritizes Environmental Compounds with Adverse Cardiovascular Activities.

Cardiovascular diseases are the leading cause of premature death in humans and remain a global public health challenge. While age, sex, family history, and false nutrition make a contribution, our understanding of compounds acting as cardiovascular disruptors is far from complete. Here, we aim to identify cardiovascular disruptors via a reduced transcriptome atlas (RTA) approach, which integrates large-scale transcriptome data sets of zebrafish and compiles a specific gene panel related to cardiovascular diseases. Among 767 gene expression profiles covering 81 environmental compounds, 11 priority compounds are identified with the greatest effects on the cardiovascular system at the transcriptional level. Among them, metals (AgNO3, Ag nanoparticles, arsenic) and pesticides/biocides (linuron, methylparaben, triclosan, and trimethylchlorotin) are identified with the most significant effects. Distinct transcriptional signatures are further identified by the percentage values, indicating that different physiological endpoints exist among prioritized compounds. In addition, cardiovascular dysregulations are experimentally confirmed for the prioritized compounds via alterations of cardiovascular physiology and lipid profiles of zebrafish. The accuracy rate of experimental verification reaches up to 62.9%. The web-based RTA analysis tool, Cardionet, for rapid cardiovascular disruptor discovery was further provided at http://www.envh.sjtu.edu.cn/cardionet.jsp. Our integrative approach yields an efficient platform to discover novel cardiovascular-disrupting chemicals in the environment.

Prednisolone Accelerates Embryonic Development of Zebrafish via Glucocorticoid Receptor Signaling at Low Concentrations.

Synthetic glucocorticoids have been widely detected in aquatic ecosystems and may pose a toxicological risk to fish. In the present study, we described multiple end point responses of zebrafish to a commonly prescribed glucocorticoid, prednisolone (PREL), at concentrations between 0.001 and 9.26 µg/L. Of 23 end points monitored, 7 were affected significantly. Significant increases in the frequency of yolk extension formation, spontaneous contraction, heart rate, and ocular melanin density and significant decreases of ear-eye distance at PREL concentrations of 0.001 µg/L and above clearly pointed to the acceleration of embryonic development of zebrafish by PREL. Further confirmation came from the alterations in somite numbers, head-trunk angle, and yolk sac size, as well as outcomes obtained via RNA sequencing, in which signaling pathways involved in tissue/organ growth and development were highly enriched in embryos upon PREL exposure. In addition, the crucial role of glucocorticoid receptor (GR) for PREL-induced effects was confirmed by both, the coexposure to antagonist mifepristone (RU486) and GR-/- mutant zebrafish experiments. We further demonstrated similar accelerations of embryonic development of zebrafish upon exposure to 11 additional glucocorticoids, indicating generic adverse effect characteristics. Overall, our results revealed developmental alterations of PREL in fish embryos at low concentrations and thus provided novel insights into the understanding of the potential environmental risks of glucocorticoids.

Large-Scale Transcriptional Profiling of Molecular Perturbations Reveals Cell Line Specific Responses and Implications for Environmental Screening.

Cell-based bioassays represent nearly half of all high-throughput screens currently conducted for risk assessment of environmental chemicals. However, there has long been a concern about the sensitivity and heterogeneity among cell lines, which were explored only in a limited manner. Here, we address this question by conducting a large-scale transcriptome analysis of the responses of discrete cell lines to specific molecules. We report the collections of >223 300 gene expression profiles from a wide array of cell lines exposed to 2243 compounds. Our results demonstrate distinct responses among cell lines at both the gene and the pathway levels. Temporal variations for a very large proportion of compounds occur as well. High sensitivity and/or heterogeneity is either cell line-specific or universal depending on the modes of action of the compounds. Among 12 representative pathways analyzed, distinct cell-chemical interactions exist. On one hand, lung carcinoma cells are always best suited for glucocorticoid receptor agonist identification, while on the other hand, high sensitivity and heterogenic features are universal for histone deacetylase inhibitors and ATPase inhibitors. Our data provide novel insights into the understanding of cell-specific responses and interactions between cells and xenobiotics. The findings have substantial implications for the design, execution, and interpretation of high-throughput screening assays in (eco)toxicology.

ABC transporters in gills of rainbow trout (Oncorhynchus mykiss).

Fish gills are a structurally and functionally complex organ at the interface between the organism and the aquatic environment. Gill functions include the transfer of organic molecules, both natural ones and xenobiotic compounds. Whether the branchial exchange of organic molecules involves active transporters is currently not known. Here, we investigated the presence, diversity and functional activity of ATP-binding cassette (ABC) transporters in gills of juvenile rainbow trout. By means of RT-qPCR, gene transcripts of members from the abcb, abcc and abcg subfamilies were identified. Comparisons with mRNA profiles from trout liver and kidney revealed that ABC transporters known to have an apical localization in polarized epithelia, especially abcc2 and abcb1, were under-represented in the gills. In contrast, ABC transporters with mainly basolateral localization showed comparable gene transcript levels in the three organs. The most prominent ABC transporter in gills was an abcb subfamily member, which was annotated as abcb5 based on the synteny and phylogeny. Functional in vivo assays pointed to a role of branchial ABC transporters in branchial solute exchange. We further assessed the utility of primary gill cell cultures to characterize transporter-mediated branchial exchange of organic molecules, by examining ABC transporter gene transcript patterns and functional activity in primary cultures. The gill cultures displayed functional transport activity, but the ABC mRNA expression patterns were different to those of the intact gills. Overall, the findings of this study provide evidence for the presence of functional ABC transporter activity in gills of fish.

Physiological and Transcriptional Effects of Mixtures of Environmental Estrogens, Androgens, Progestins, and Glucocorticoids in Zebrafish.

Fishes are exposed to mixtures of different classes of steroids, but ecotoxicological implications are not sufficiently known. Here, we systematically analyze effects of different combinations of steroid mixtures in zebrafish embryos to assess their joint activities on physiology and transcriptional alterations of steroid-specific target genes at 96 and 120 h post fertilization. In binary mixtures of clobetasol propionate (CLO) with estradiol (E2) or androstenedione (A4), each steroid exhibited its own expression profile. This was also the case in mixtures of 5-, 8-, and 13-different classes of steroids in exposure concentrations of 10-10,000 ng/L. The transcriptional expression of most genes in different mixtures was steroid-specific except for genes encoding aromatase (cyp19b), sulfotransferase (sult2st3), and cyp2k22 that were induced by androgens, progestins, and glucocorticoids. Marked alterations occurred for sult2st3 in binary mixtures of CLO + E2 and CLO + A4. Glucocorticoids increased the heart rate and muscle contractions. In mixtures containing estrogens, induction of the cyp19b transcript occurred at 10 ng/L and protc from the anticoagulation system at 100 ng/L. Our study demonstrates that steroids can act independently in mixtures; the sum of individual steroid profiles is expressed. However, some genes, including cyp19b, sult2st3, and cyp2k22, are regulated by several steroids. This joint effect on different pathways may be of concern for fish development.

Mixtures of Aluminum and Indium Induce More than Additive Phenotypic and Toxicogenomic Responses in Daphnia magna.

Aquatic systems are contaminated by many metals but their effects as mixtures on organisms are not well understood. Here, we assessed effects of aluminum with fairly well-known modes of actions and indium, an understudied emerging contaminant from electronics, followed by studying equi-effective mixtures thereof. We report acute and adverse phenotypic effects in Daphnia magna adults and global transcriptomic effects employing RNA sequencing in neonates. The mixture induced more than additive activity in mortality and in physiological effects, including growth and reproduction. Similarly, transcriptomic effects were more than additive, as indicated by a markedly higher number of 463 differentially expressed transcripts in the mixture and by distinct classes of genes assigned to several biological functions, including metabolic processes, suggesting depleted energy reserves, which may be responsible for the observed impaired reproduction and growth. A gene set enrichment analysis (GSEA) of a priori known response pathways for aluminum confirmed activation of distinct molecular pathways by indium. Our study is highlighting more than additive effects at the transcriptional and physiological level and is providing a state-of-the art approach to mixture analysis, which is important for risk assessment of these metals and metal mixtures.

Active Glucocorticoids Have a Range of Important Adverse Developmental and Physiological Effects on Developing Zebrafish Embryos.

Glucocorticoids in aquatic systems originating from natural excretion and medical use may pose a risk to fish. Here, we analyzed physiological and transcriptional effects of clobetasol propionate (CLO), cortisol and cortisone in zebrafish embryos as single compounds and binary mixtures. CLO and cortisol, but not cortisone showed a concentration-dependent decrease in muscle contraction, increase in heart rate, and accelerated hatching. CLO also induced immobilization and edema at high concentrations. Transcription analysis covering up to 26 genes showed that mostly genes related to glucose metabolism, immune system and development were differentially expressed at 91 ng/L and higher. CLO showed stronger effects on immune system genes than cortisol, which was characterized by upregulation of fkbp5, irg1l, gilz, and socs3, and development genes, matrix metalloproteinases mmp-9 and mmp-13, while cortisol led to stronger upregulation of the gluconeogenesis genes g6pca and pepck1. CLO also induced genes regulating the circadian rhythm, nr1d1 and per1a. In contrast, cortisone led to down-regulation of vitellogenin. Binary mixtures of cortisol and CLO mostly showed a similar activity as CLO alone on physiological and transcriptional end points but additive effects in heart rate and pepck1 upregulation, which indicates that mixtures of glucocorticoids may be of concern for developing fish.

Global Transcriptomic Effects of Environmentally Relevant Concentrations of the Neonicotinoids Clothianidin, Imidacloprid, and Thiamethoxam in the Brain of Honey Bees ( Apis mellifera).

Neonicotinoids are implicated in the decline of honey bees, but the molecular basis underlying adverse effects is poorly known. Here we describe global transcriptomic profiles in the brain of honey bee workers exposed for 48 h at one environmentally realistic and one sublethal concentration of 0.3 and 3.0 ng/bee clothianidin and imidacloprid, respectively, and 0.1 and 1.0 ng/bee thiamethoxam (1-30 ng/mL sucrose solution) by high-throughput RNA-sequencing (RNA-seq). All neonicotinoids led to significant alteration (mainly down-regulation) of gene expression, generally with a concentration-dependent effect. Among many others, genes related to metabolism and detoxification were differently expressed. Gene ontology (GO) enrichment analysis of biological processes revealed catabolic carbohydrate metabolism (regulation of enzyme activities such as amylase), lipid metabolism, and transport mechanisms as shared terms between all neonicotinoids at high concentrations. KEGG pathway analysis indicated that at least two neonicotinoids induced changes in expression of various metabolic pathways: pentose phosphate pathways, starch and sucrose metabolism, and sulfur metabolism, in which glucose 1-dehydrogenase and alpha-amylase were down-regulated and 3'(2'), 5'-bisphosphate nucleotidase was up-regulated. RT-qPCR analysis confirmed the down-regulation of major royal jelly proteins, hbg3, and cyp9e2 found by RNA-seq. Our study highlights the comparative molecular effects of neonicotinoid exposure to bees. Further studies should link these effects with physiological outcomes for a better understanding of effects of neonicotinoids.

Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2ß, gap-43, neurofilament-h, tubulin-α and tubulin-ß. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals.

Occurrence and Ecotoxicological Effects of Free, Conjugated, and Halogenated Steroids Including 17α-Hydroxypregnanolone and Pregnanediol in Swiss Wastewater and Surface Water.

Apart from estrogens, the occurrence and ecotoxicity of steroids in aquatic environments is poorly known. Here, we analyzed 33 steroids, including estrogens, androgens, progestins, and glucocorticoids, in hospital wastewaters, river water, and municipal wastewater treatment plant (WTP) influents and effluents at different sites in Switzerland. In addition, wastewater from different treatment steps of two WTPs with advanced treatment, such as ozonation or pulverized activated carbon, were analyzed to study the steroid's behavior during treatment. Considerable levels of different steroids occurred in hospital and raw municipal wastewater, but they were low (lower than 1 ng/L) or below the detection level in effluents of WTPs and river water. In WTP influents, estrogens (estrone, 17ß-estradiol, and estriol), androgens (androstenedione, androsterone, trans-androsterone, and testosterone), progestins and metabolites (progesterone, medroxyprogesterone acetate, megestrol acetate, mifepristone, pregnanediol, 17α-hydroxypregnanolone, 17α-hydroxyprogesterone, and 21α-hydroxyprogesterone) were detected and removed effectively during biological treatment. Ozonation further removed the steroids. Exposure of zebrafish embryos demonstrated negligible effects of pregnanediol and 17α-hydroxypregnanolone, while mixtures that mimic wastewater and river water composition affected embryo development and led to the alteration of steroidogenesis gene transcripts at nanogram per liter concentrations. Although steroid concentrations are low in Swiss rivers, the possibility of additive effects may be of concern.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1µM) and toxic concentrations (5µM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5µM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10µM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin.

Time Matters in Ecotoxicological Sampling Due to Circadian Rhythm.

As in general, technological inventions also drive the development in the field of toxicology and ecotoxicology. In the past decade, gene expression analysis has become a universally applied technology allowing many insights into toxicological pathways of environmental contaminants. Due to the novel technologies, including quantitative determination of mRNA by quantitative reverse transcription analysis (qRT-PCR), and semiquantitative methods, such as microarrays and RNA-sequencing technologies, toxicological profiles of contaminants could be identified. For instance, gene expression analysis of genes associated with the hypothalamic-pituitary-gonadal axis (HPG axis) in fish had become a conventional end point for endocrine disrupting chemicals. While these gene expression data provide novel insights into identifying potential toxicological end points and molecular mechanisms, often not enough attention is given to the question of mRNA stabilities and reliabilities of transcriptional data, in particular when links to physiological effects are difficult to make. A crucial factor in this issue is the endogenous circadian oscillations of genes during sampling.

Molecular Effects of Neonicotinoids in Honey Bees (Apis mellifera).

Neonicotinoids are implicated in the decline of bee populations. As agonists of nicotinic acetylcholine receptors, they disturb acetylcholine receptor signaling leading to neurotoxicity. Several behavioral studies showed the link between neonicotinoid exposure and adverse effects on foraging activity and reproduction. However, molecular effects underlying these effects are poorly understood. Here we elucidated molecular effects at environmental realistic levels of three neonicotinoids and nicotine, and compared laboratory studies to field exposures with acetamiprid. We assessed transcriptional alterations of eight selected genes in caged honey bees exposed to different concentrations of the neonicotinoids acetamiprid, clothianidin, imidacloporid, and thiamethoxam, as well as nicotine. We determined transcripts of several targets, including nicotinic acetylcholine receptor α 1 and α 2 subunit, the multifunctional gene vitellogenin, immune system genes apidaecin and defensin-1, stress-related gene catalase and two genes linked to memory formation, pka and creb. Vitellogenin showed a strong increase upon neonicotinoid exposures in the laboratory and field, while creb and pka transcripts were down-regulated. The induction of vitellogenin suggests adverse effects on foraging activity, whereas creb and pka down-regulation may be implicated in decreased long-term memory formation. Transcriptional alterations occurred at environmental concentrations and provide an explanation for the molecular basis of observed adverse effects of neonicotinoids to bees.

Corticosteroid Fludrocortisone Acetate Targets Multiple End Points in Zebrafish (Danio rerio) at Low Concentrations.

Synthetic corticosteroids may pose an environmental risk to fish. Here, we describe multiend point responses of adult zebrafish (8 months old) upon 21-day exposure to a commonly prescribed corticosteroid, fludrocortisone acetate (FLU), at concentrations between 0.006 and 42 µg/L. No remarkable reproductive impacts were observed, while physiological effects, including plasma glucose level and blood leukocyte numbers were significant altered even at 42 ng/L. Ovary parameters and transcriptional analysis of hypothalamic-pituitary-gonadal-liver axis revealed negligible effects. Significant alterations of the circadian rhythm network were observed in the zebrafish brain. Transcripts of several biomarker genes, including per1a and nr1d1, displayed strong transcriptional changes, which occurred at environmental relevant concentrations of 6 and 42 ng/L FLU. Importantly, the development and behavior of F1 embryos were significant changed. Heartbeat, hatching success and swimming behavior of F1 embryos were all increased even at 6 and 42 ng/L. All effects were further confirmed by exposure of eleuthero-embryos. Significant transcriptional changes of biomarker genes involved in gluconeogenesis, immune response and circadian rhythm in eleuthero-embryos confirmed the observations in adult fish. Hatching success, heartbeat, and swimming activity were increased at 81 ng/L and higher, as with F1 embryos. These results provide novel insights into the understanding of potential environmental risks of corticosteroids.

Preparation of Fluorescent Microcystin Derivatives by Direct Arginine Labelling and Their Biological Evaluation.

Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract.

Environmental Progestins Progesterone and Drospirenone Alter the Circadian Rhythm Network in Zebrafish (Danio rerio).

Progestins alter hormone homeostasis and may result in reproductive effects in humans and animals. Thus far, studies in fish have focused on the hypothalamic-pituitary-gonadal (HPG)-axis and reproduction, but other effects have little been investigated. Here we report that progesterone (P4) and drospirenone (DRS) interfere with regulation of the circadian rhythm in fish. Breeding pairs of adult zebrafish were exposed to P4 and DRS at concentrations between 7 and 13 650 ng/L for 21 days. Transcriptional analysis revealed significant and dose-dependent alterations of the circadian rhythm network in the brain with little effects in the gonads. Significant alterations of many target transcripts occurred even at environmental relevant concentrations of 7 ng/L P4 and at 99 ng/L DRS. They were fully consistent with the well-described circadian rhythm negative/positive feedback loops. Transcriptional alterations of the circadian rhythm network were correlated with those in the HPG-Liver-axis. Fecundity was decreased at 742 (P4) and 2763 (DRS) ng/L. Dose-dependent alterations in the circadian rhythm network were also observed in F1 eleuthero-embryos. Our results suggest a potential target of environmental progestins, the circadian rhythm network, in addition to the adverse reproductive effects. Forthcoming studies should show whether the transcriptional alterations in circadian rhythm translate into physiological effects.

Synthetic progestins medroxyprogesterone acetate and dydrogesterone and their binary mixtures adversely affect reproduction and lead to histological and transcriptional alterations in zebrafish (Danio rerio).

Medroxyprogesterone acetate (MPA) and dydrogesterone (DDG) are synthetic progestins widely used in human and veterinary medicine. Although aquatic organisms are exposed to them through wastewater and animal farm runoff, very little is known about their effects in the environment. Here we provide a comprehensive analysis of the responses of zebrafish (Danio rerio) to MPA, DDG, and their binary mixtures at measured concentrations between 4.5 and 1663 ng/L. DDG and both mixtures impaired reproductive capacities (egg production) of breeding pairs and led to histological alterations of ovaries and testes and increased gonadosomatic index. Transcriptional analysis of up to 28 genes belonging to different pathways demonstrated alterations in steroid hormone receptors, steroidogenesis enzymes, and specifically, the circadian rhythm genes, in different organs of adult zebrafish and eleuthero-embryos. Alterations occurred even at environmentally relevant concentrations of 4.5-4.8 ng/L MPA, DDG and the mixture in eleuthero-embryos and at 43-89 ng/L in adult zebrafish. Additionally, the mixtures displayed additive effects in most but not all parameters in adults and eleuthero-embryos, suggesting concentration addition. Our data suggest that MPA and DDG and their mixtures induce multiple transcriptional responses at environmentally relevant concentrations and adverse effects on reproduction and gonad histology at higher levels.

Molecular characterization of zebrafish Oatp1d1 (Slco1d1), a novel organic anion-transporting polypeptide.

The organic anion-transporting polypeptide (OATP/Oatp) superfamily includes a group of polyspecific transporters that mediate transport of large amphipathic, mostly anionic molecules across cell membranes of eukaryotes. OATPs/Oatps are involved in the disposition and elimination of numerous physiological and foreign compounds. However, in non-mammalian species, the functional properties of Oatps remain unknown. We aimed to elucidate the role of Oatp1d1 in zebrafish to gain insights into the functional and structural evolution of the OATP1/Oatp1 superfamily. We show that diversification of the OATP1/Oatp1 family occurs after the emergence of jawed fish and that the OATP1A/Oatp1a and OATP1B/Oatp1b subfamilies appeared at the root of tetrapods. The Oatp1d subfamily emerged in teleosts and is absent in tetrapods. The zebrafish Oatp1d1 is similar to mammalian OATP1A/Oatp1a and OATP1B/Oatp1b members, with the main physiological role in transport and balance of steroid hormones. Oatp1d1 activity is dependent upon pH gradient, which could indicate bicarbonate exchange as a mode of transport. Our analysis of evolutionary conservation and structural properties revealed that (i) His-79 in intracellular loop 3 is conserved within OATP1/Oatp1 family and is crucial for the transport activity; (ii) N-glycosylation impacts membrane targeting and is conserved within the OATP1/Oatp1 family with Asn-122, Asn-133, Asn-499, and Asn-512 residues involved; (iii) the evolutionarily conserved cholesterol recognition interaction amino acid consensus motif is important for membrane localization; and (iv) Oatp1d1 is present in dimeric and possibly oligomeric form in the cell membrane. In conclusion, we describe the first detailed characterization of a new Oatp transporter in zebrafish, offering important insights into the functional evolution of the OATP1/Oatp1 family and the physiological role of Oatp1d1.

Additive and synergistic antiandrogenic activities of mixtures of azol fungicides and vinclozolin.

OBJECTIVE: Many pesticides including pyrethroids and azole fungicides are suspected to have an endocrine disrupting property. At present, the joint activity of compound mixtures is only marginally known. Here we tested the hypothesis that the antiandrogenic activity of mixtures of azole fungicides can be predicted by the concentration addition (CA) model. METHODS: The antiandrogenic activity was assessed in MDA-kb2 cells. Following assessing single compounds activities mixtures of azole fungicides and vinclozolin were investigated. Interactions were analyzed by direct comparison between experimental and estimated dose-response curves assuming CA, followed by an analysis by the isobole method and the toxic unit approach. RESULTS: The antiandrogenic activity of pyrethroids deltamethrin, cypermethrin, fenvalerate and permethrin was weak, while the azole fungicides tebuconazole, propiconazole, epoxiconazole, econazole and vinclozolin exhibited strong antiandrogenic activity. Ten binary and one ternary mixture combinations of five antiandrogenic fungicides were assessed at equi-effective concentrations of EC25 and EC50. Isoboles indicated that about 50% of the binary mixtures were additive and 50% synergistic. Synergism was even more frequently indicated by the toxic unit approach. CONCLUSION: Our data lead to the conclusion that interactions in mixtures follow the CA model. However, a surprisingly high percentage of synergistic interactions occurred. Therefore, the mixture activity of antiandrogenic azole fungicides is at least additive. PRACTICE: Mixtures should also be considered for additive antiandrogenic activity in hazard and risk assessment. IMPLICATIONS: Our evaluation provides an appropriate "proof of concept", but whether it equally translates to in vivo effects should further be investigated.