Dibutyl phthalate disrupts energy metabolism and morphology in the gills and induces hepatotoxicity in zebrafish.

This study investigated the acute effects of dibutyl phthalate (DBP) exposure on energy metabolism and gill histology in zebrafish (Danio rerio). The in vitro incubation of gill tissue with 10 µM DBP for 60 min altered tissue energy supply, as shown by decreased lactate content and lactate dehydrogenase (LDH) activity. Higher concentrations of DBP (100 µM and 1 mM) increased lactate content and LDH activity; however, they blocked glucose uptake, depleted the glycogen content in cellular stores, and induced injury to the gills, as measured by LDH release to the extracellular medium. In addition, in vivo exposure of fish to 1 pM DBP for 12 h induced liver damage by increasing alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT) activities. Gill histology indicated hyperemia, lamellar fusion, lamellar telangiectasis, and necrosis. Data indicate that acute exposure of zebrafish gills to the higher DBP concentrations studied induces anaerobic cellular activity and high lactate production, causing gill damage, diminishing cell viability, and incurring liver dysfunction.

Influence of the aquatic environment and 1α,25(OH)2 vitamin D3 on calcium influx in the intestine of adult zebrafish.

We investigated the effects of environment calcium challenge and 1α,25(OH)2 vitamin D3 (1,25-D3) on 45Ca2+ influx in the intestine of zebrafish (ZF). In vitro45Ca2+ influx was analyzed using intestines from fed and fasted fish. ZF were held in water containing Ca2+ (0.02, 0.7, 2.0 mM) to analyze the ex vivo45Ca2+ influx in the intestine and for histology. Intestines from fish held in water with Ca2+ were incubated ex vivo to characterize ion channels, receptors, ATPases and ion exchangers that orchestrate 45Ca2+ influx. For in vitro studies, intestines were incubated with antagonists/agonist or inhibitors to study the mechanism of 1,25-D3 on 45Ca2+ influx. Fasted ZF reached a plateau for 45Ca2+ influx at 30 min. In vivo fish at high Ca2+ stimulated ex vivo45Ca2+ influx and increased the height of intestinal villi in low calcium. In the normal calcium, 45Ca2+ influx was maintained by the reverse-mode Na+/Ca2+ (NCX) activation, Na+/K+-ATPase pump and sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. However, Ca2+ hyperosmolarity is supported by L-type voltage-dependent calcium channels (L-VDCC), transient receptor potential vanilloid subfamily 1 (TRPV1) and Na+/K+-ATPase activity. The calcium challenge causes morphological alteration and changes the ion type-channels involved in the intestine to maintain hyperosmolarity. 1,25-D3 stimulates Ca2+ influx in normal osmolarity coordinated by L-VDCC activation and SERCA inhibition to keeps high intracellular calcium in intestine. Our data showed that the adult ZF regulates the calcium challenge (per se osmolarity), independently of the hormonal regulation to maintain the calcium balance through the intestine to support ionic adaptation.

Effect of water temperature and food availability on growth performance, sex ratio and gonadal development in juvenile convict cichlid (Amatitlanianigrofasciata).

Temperature and food availability are key drivers of growth and reproductive development in fishes, but information on how they interact is poorly understood. This study investigates the effects of water temperature and food availability on growth, sex ratio and gonadal development of the convict cichlid (Amatitlania nigrofasciata) which is an ornamental fish that may be a useful lab model. For this experiment, 180 juvenile convict cichlid (0.3 ± 0.02 g) were held at three different temperatures (26, 29 and 32 °C as T1, T2 and T3) and fed to satiation (S) or a restricted diet (R: half satiation) during a 56-day experimental period. Specific growth rate was significantly higher in T2S treatment than the other groups. The highest and lowest mean oocyte sizes were recorded in T1S and T3R groups, respectively. The sex ratio of fish held at 29 °C was male biased (female, 21.0%; male, 78.9%), but this was not seen at 26 °C (female, 47.6%; male, 52.4%) or 32 °C (female, 57.1%; male, 42.9%). In T1S and T1R treatments, oocytes developed more than the other treatments and in T2S group testicular development was more advanced than other groups. These results demonstrate the complex interplay of temperature and food availability on growth and reproductive development in the convict cichlid. Appropriate food availability significantly improves growth and reproductive processes, while restricted feeding decreases growth, survival rate and reproductive performance.

Calcium influx and spermatogenesis in the testis and liver enzyme activities in the zebrafish are rapidly modulated by the calcium content of the water.

This study investigated the effects of varying environmental Ca2+ concentrations on the influx of Ca2+ to the testis, testicular morphology, and liver enzymes in the zebrafish. Adult zebrafish (Danio rerio) were held in water containing low (0.02 mM), control (0. 7 mM) or high (2 mM) Ca2+ concentrations for 12 h. Testes were then incubated in vitro with 0.1 µCi/mL 45Ca2+ to measure Ca2+ influx at 30 and 60 min and qualitative and quantitative testicular histological analyses were conducted. In addition, activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT), enzymes that indicate tissue damage, were evaluated in the liver. The testes from zebrafish exposed in vivo to low (0.02 mM) and high (2 mM) Ca2+ content water had a higher Ca2+ influx than the control group after 30 min of incubation, and at 60 min (high Ca2+ group only). There were morphological changes in the testes from the low and high Ca2+ groups including spermatozoa distributed in dense agglomerates and apoptotic cells. Furthermore, zebrafish exposed to high Ca2+ containing water had an increased density of haploid cells (spermatids and spermatozoa). In addition, both low and high Ca2+ water affected liver function by increasing ALT and GGT activities. Collectively, these studies show that alterations in calcium homeostasis in the testis, stimulation of the spermatogenic wave and hepatic injury were rapid responses to changes in the concentration of Ca2+ in the water.

In vivo and in vitro short-term bisphenol A exposures disrupt testicular energy metabolism and negatively impact spermatogenesis in zebrafish.

This study investigated the in vitro and short-term in vivo effects of Bisphenol A (BPA) on testicular energy metabolism and morphology in the zebrafish (Danio rerio). Testes were incubated in vitro for 1 h or fish were exposed in vivo to BPA in the tank water for 12 h. Testicular lactate, glycogen and cholesterol were measured and 14C-deoxy-d-glucose uptake and activity of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. In addition, testis samples from the in vivo exposures were subject to digital analysis of testicular cells using Ilastik software and the Pixel Classification module and estimation of apoptosis by Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) immunohistochemical analysis. Our results from in vitro studies showed that BPA at 10 pM and 10 µM decreased testicular lactate content, glycogen content and LDH activity, but increased testicular AST activity. In addition, only BPA at 10 pM significantly decreased testicular ALT activity and cholesterol content. However, 14C-deoxy-d-glucose uptake was not changed. Furthermore, our results from in vivo studies showed that 10 pM BPA but not 10 µM BPA reduced testicular content of lactate and glycogen. In addition, both BPA concentrations decreased AST activity, whereas only BPA at 10 µM reduced ALT activity. However, LDH activity was not changed. Additionally, both concentrations of BPA induced spermatocyte apoptosis and a decrease in the proportion of the surface area of spermatids and spermatozoa. Collectively these data suggest that short-term BPA exposure affects energy metabolism and spermatogenesis in male zebrafish.

Nuclear progesterone receptor regulates ptger4b and PLA2G4A expression in zebrafish (Danio rerio) ovulation.

Previous studies have implicated the nuclear progesterone receptor (Pgr or nPR) as being critical to ovulation in fishes. This study investigated the expression of Pgr in zebrafish ovarian follicles throughout development as well as putative downstream targets of Pgr by searching the promoter regions of selected genes for specific DNA sequences to which Pgr binds and acts as a transcription factor. Expression of Pgr mRNA increases dramatically as follicles grow and mature. In silico analysis of selected genes linked to ovulation showed that the prostaglandin receptors ptger4a and ptger4b contained the progesterone responsive element (PRE) GRCCGGA in their promoter regions. Studies using full-grown follicles incubated in vitro revealed that ptger4b was upregulated in response to 17,20ß-P. Our studies also showed that the expression of phospholipase A2 (PLA2G4A) mRNA and protein, a key enzyme in prostaglandin synthesis, was upregulated in response to 17,20ß-P treatment. pla2g4a was not found to contain a PRE, indicating that it is regulated indirectly by 17,20ß-P or that it may contain an as-of-yet unidentified PRE in its promoter region. Collectively, these studies provide further evidence of the importance of Pgr during the periovulatory periods through its involvement in prostaglandin production and function by controlling expression of PLA2G4A and the receptor EP4b and that these genes appear to be regulated through the actions of 17,20ß-P.

Assessment of risks to listed species from the use of atrazine in the USA: a perspective.

Atrazine is a triazine herbicide used predominantly on corn, sorghum, and sugarcane in the US. Its use potentially overlaps with the ranges of listed (threatened and endangered) species. In response to registration review in the context of the Endangered Species Act, we evaluated potential direct and indirect impacts of atrazine on listed species and designated critical habitats. Atrazine has been widely studied, extensive environmental monitoring and toxicity data sets are available, and the spatial and temporal uses on major crops are well characterized. Ranges of listed species are less well-defined, resulting in overly conservative designations of "May Effect". Preferences for habitat and food sources serve to limit exposure among many listed animal species and animals are relatively insensitive. Atrazine does not bioaccumulate, further diminishing exposures among consumers and predators. Because of incomplete exposure pathways, many species can be eliminated from consideration for direct effects. It is toxic to plants, but even sensitive plants tolerate episodic exposures, such as those occurring in flowing waters. Empirical data from long-term monitoring programs and realistic field data on off-target deposition of drift indicate that many other listed species can be removed from consideration because exposures are below conservative toxicity thresholds for direct and indirect effects. Combined with recent mitigation actions by the registrant, this review serves to refine and focus forthcoming listed species assessment efforts for atrazine.Abbreviations: a.i. = Active ingredient (of a pesticide product). AEMP = Atrazine Ecological Monitoring Program. AIMS = Avian Incident Monitoring SystemArach. = Arachnid (spiders and mites). AUC = Area Under the Curve. BE = Biological Evaluation (of potential effects on listed species). BO = Biological Opinion (conclusion of the consultation between USEPA and the Services with respect to potential effects in listed species). CASM = Comprehensive Aquatic System Model. CDL = Crop Data LayerCN = field Curve Number. CRP = Conservation Reserve Program (lands). CTA = Conditioned Taste Avoidance. DAC = Diaminochlorotriazine (a metabolite of atrazine, also known by the acronym DACT). DER = Data Evaluation Record. EC25 = Concentration causing a specified effect in 25% of the tested organisms. EC50 = Concentration causing a specified effect in 50% of the tested organisms. EC50RGR = Concentration causing a 50% reduction in relative growth rate. ECOS = Environmental Conservation Online System. EDD = Estimated Daily Dose. EEC = Expected Environmental Concentration. EFED = Environmental Fate and Effects Division (of the USEPA). EFSA = European Food Safety Agency. EIIS = Ecological Incident Information System. ERA = Environmental Risk Assessment. ESA = Endangered Species Act. ESU = Evolutionarily Significant UnitsFAR = Field Application RateFIFRA = Federal Insecticide, Fungicide, and Rodenticide Act. FOIA = Freedom of Information Act (request). GSD = Genus Sensitivity Distribution. HC5 = Hazardous Concentration for ≤ 5% of species. HUC = Hydrologic Unit Code. IBM = Individual-Based Model. IDS = Incident Data System. KOC = Partition coefficient between water and organic matter in soil or sediment. KOW = Octanol-Water partition coefficient. LC50 = Concentration lethal to 50% of the tested organisms. LC-MS-MS = Liquid Chromatograph with Tandem Mass Spectrometry. LD50 = Dose lethal to 50% of the tested organisms. LAA = Likely to Adversely Affect. LOAEC = Lowest-Observed-Adverse-Effect Concentration. LOC = Level of Concern. MA = May Affect. MATC = Maximum Acceptable Toxicant Concentration. NAS = National Academy of Sciences. NCWQR = National Center of Water Quality Research. NE = No Effect. NLAA = Not Likely to Adversely Affect. NMFS = National Marine Fisheries Service. NOAA = National Oceanic and Atmospheric Administration. NOAEC = No-Observed-Adverse-Effect Concentration. NOAEL = No-Observed-Adverse-Effect Dose-Level. OECD = Organization of Economic Cooperation and Development. PNSP = Pesticide National Synthesis Project. PQ = Plastoquinone. PRZM = Pesticide Root Zone Model. PWC = Pesticide in Water Calculator. QWoE = Quantitative Weight of Evidence. RGR = Relative growth rate (of plants). RQ = Risk Quotient. RUD = Residue Unit Doses. SAP = Science Advisory Panel (of the USEPA). SGR = Specific Growth Rate. SI = Supplemental Information. SSD = Species Sensitivity Distribution. SURLAG = Surface Runoff Lag Coefficient. SWAT = Soil & Water Assessment Tool. SWCC = Surface Water Concentration Calculator. UDL = Use Data Layer (for pesticides). USDA = United States Department of Agriculture. USEPA = United States Environmental Protection Agency. USFWS = United States Fish and Wildlife Service. USGS = United States Geological Survey. WARP = Watershed Regressions for Pesticides.

ADAMTS1 is regulated by the EP4 receptor in the zebrafish ovary.

Prostaglandins (PGs) are a class of fatty-acid derived hormones that are essential in ovulation of teleosts, but their exact role remains unknown. One putative target of PGs in ovulation is regulation of the expression of members of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family, which are implicated in follicular rupture. This study investigated the regulation of ADAMTS, other proteases, and their inhibitors in response to treatment with PGE2 or PGF2α. Four members of the ADAMTS family, ADAMTS1, ADAMTS5, ADAMTS9, and ADAMTS16 were shown to be expressed in the ovary of zebrafish, but only adamts1 was upregulated in full-grown follicles following treatment with PGE2. Inhibitors of the PG receptors EP1 and EP2 had no effect on PGE2-stimulated adamts1 expression, while treatment of full-grown follicles with both PGE2 and GW627368x, an inhibitor of EP4 function, prevented the PGE2-induced increase in adamts1 expression. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) in vitro had no effect on the expression of adamts1 mRNA. These findings suggest that expression of ADAMTS1 in zebrafish ovarian follicles is regulated by the prostaglandin PGE2 via the EP4 series prostaglandin receptor.

Triterpene betulin may be involved in the acute effects of pulp and paper mill effluent on testis physiology in zebrafish.

Pulp and paper mill effluent can cause changes in the morphology and energy metabolism in the zebrafish (Danio rerio) testis. Betulin, a naturally occurring triterpene is commonly present in this type of effluent and is suspected of being involved in these effects. The aim of this study was to compare the effects pulp and paper mill effluent and betulin on various aspects of testicular physiology in the zebrafish. This included the in vitro effects of effluent and betulin on testicular lactate content and lactate dehydrogenase (LDH) activity. In addition, the effects of betulin on glucose uptake, glycogen, alanine aminotransferase (ALT), reactive oxygen and nitrogen species formation and oxidative damage in the testes were determined. Furthermore, we compared the effects and mechanism of action of betulin and effluent on calcium homeostasis in testes. In vitro exposure to both effluent and betulin decreased lactate and calcium influx, possibly due to the activation of the sodium­calcium exchanger (NCX) pump. Additionally, betulin-treated testes had higher reactive oxygen species (ROS) and reduced glutathione (GSH) content, as well as increased glutathione transferase (GST) activity and a tendency towards decreased catalase (CAT) activity. Thus, this study shows that alterations in testis physiology caused by the pulp and paper mill effluent in the testis may be due in part to the actions of betulin.

Pyriproxyfen induces intracellular calcium overload and alters antioxidant defenses in Danio rerio testis that may influence ongoing spermatogenesis.

We investigated the in vitro effects of pyriproxyfen on ionic balance in the testis of the zebrafish by measuring 45Ca2+ influx. In vivo pyriproxyfen treatment was carried out to study oxidative stress, and conduct morphological analysis of the testis and liver. Whole testes were incubated in vitro with/without pyriproxyfen (10-12, 10-9 or 10-6 M; 30 min) and 45Ca2+ influx determined. To study pyriproxyfen's mechanism of action, inhibitors/activators of ionic channels or pumps/exchangers, protein kinase inhibitors or a calcium chelator were added 15 min before the addition of 45Ca2+ and pyriproxyfen. We evaluated the in vivo effects of 7 day exposure to waterborne pyriproxyfen (10-9 M) on reactive oxygen species (ROS) formation, lipid peroxidation, and reduced glutathione content (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and γ-glutamyltransferase (GGT) activity. Morphological analyses of the testis and liver were carried out after in vivo exposure of D. rerio to pyriproxyfen. Pyriproxyfen increased 45Ca2+ influx by opening the voltage-dependent T-type channels (T-type VDCC), inhibiting sarco/endoplasmic reticulum 45Ca2+-ATPase (SERCA) and the NCX exchanger (forward mode) and by mobilizing calcium from stores. The involvement of potassium channels and protein kinase C (PKC) was also demonstrated in pyriproxyfen-induced intracellular calcium elevation. In vivo pyriproxyfen treatment of D. rerio increased lipid peroxidation, decreased GSH content and increased GST activity in testes, in addition to increasing the number and size of spermatogonia cysts and inducing hepatocyte basophilia and dilation of blood vessels in the liver. The toxicity of pyriproxyfen is mediated by calcium overload, increased lipid peroxidation, and a diminished antioxidant capacity in the testis, due to GSH depletion, and altered spermatogenesis. The development of high basophilia in the liver suggests that pyriproxyfen may have estrogenic activity, possibly acting as an endocrine-disruptor. These findings indicate that these alterations may contribute to pyriproxyfen toxicity and spermatogenesis disruption.

Dibutyl phthalate rapidly alters calcium homeostasis in the gills of Danio rerio.

This study investigates the impacts of exposure to an environment Ca2+ challenge and the mechanism of action of dibutyl phthalate (DBP) on Ca2+ influx in the gills of Danio rerio. In vitro profile of 45Ca2+ influx in gills was verified through the basal time-course. Fish were exposed to low, normal and high Ca2+ concentrations (0.02, 0.7 and 2 mM) for 12 h. So, gills were morphologically analysed and ex vivo45Ca2+ influx at 30 and 60 min was determined. For the in vitro studies, gills were treated for 60 min with DBP (1 pM, 1 nM and 1 µM) with/without blockers/activators of ionic channels, Ca2+ chelator, inhibitors of ATPases, ionic exchangers and protein kinase C to study the mechanism of DBP-induced 45Ca2+ influx. Exposure to high environmental Ca2+ augmented 45Ca2+ influx when compared to fish exposed to normal and low Ca2+ concentrations. Additionally, histopathological changes were observed in the gills of fish maintained for 12 h in low and high Ca2+. In vitro exposure of gills to DBP (1 pM) disturbed Ca2+ homeostasis. DBP stimulated 45Ca2+ influx in gills through the transitory receptor potential vanilloid 1 (TRPV1), and reverse-mode Na+/Ca2+ exchanger (NCX) activation, protein kinase C and K+ channels and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). These data suggest that in vivo short-term exposure of gills to low and high Ca2+ leads to 45Ca2+ influx and histopathological changes. Additionally, the DBP-induced rapid 45Ca2+ influx is mediated by TRPV1, NCX activation with the involvement of PKC, K+-channels and SERCA, thereby altering Ca2+ homeostasis.

A Brazilian pulp and paper mill effluent disrupts energy metabolism in immature rat testis and alters Sertoli cell secretion and mitochondrial activity.

Our objective was to investigate whether the pulp and paper mill industry effluent could affect the testis and Sertoli cells in a fast exposure period. For this, the present study was carried out in immature rats at 10-day-old. Testis treated in vitro with 4% effluent for 1 h presented changes in energy metabolism in terms of a decrease in lactate content and glucose uptake. Elevation in GSH content, as an antioxidant defense mechanism, was also detected. Sertoli cells treated with 4% effluent for 1 hour showed alterations in the mitochondrial metabolism that favor the decoupling of oxidative phosphorylation and the generation of oxygen reactive species and also a time and concentration-dependent delay secretion of acidic vesicles. Our results showed that pollutants present in the pulp and paper mill effluents, in a short time of exposure, are capable of inducing alterations in important metabolic functions in the testis and in Sertoli cells that are crucial for the correct progression of spermatogenesis and fertility.

Role of bisphenol A on calcium influx and its potential toxicity on the testis of Danio rerio.

This study investigated the acute in vitro effect of low-concentration bisphenol A (BPA) on calcium (45Ca2+) influx in zebrafish (Danio rerio) testis and examined whether intracellular Ca2+ was involved in the effects of BPA on testicular toxicity. In vitro studies on 45Ca2+ influx were performed in the testes after incubation with BPA for 30 min. Inhibitors were added 15 min before the addition of 45Ca2+ and BPA to testes to study the mechanism of action of BPA. The involvement of intracellular calcium from stores on lactate dehydrogenase (LDH) release and on triacylglycerol (TAG) content were carried out after in vitro incubation of testes with BPA for 1 h. Furthermore, gamma-glutamyl transpeptidase (GGT) and aspartate aminotransferase (AST) activities were analyzed in the liver at 1 h after in vitro BPA incubation of D. rerio. Our data show that the acute in vitro treatment of D. rerio testes with BPA at very low concentration activates plasma membrane ionic channels, such as voltage-dependent calcium channels and calcium-dependent chloride channels, and protein kinase C (PKC), which stimulates Ca2+ influx. In addition, BPA increased cytosolic Ca2+ by activating inositol triphosphate receptor (IP3R) and inhibiting sarco/endoplasmic reticulum calcium ATPase (SERCA) at the endoplasmic reticulum, contributing to intracellular Ca2+ overload. The protein kinases, PKC, MEK 1/2 and PI3K, are involved in the mechanism of action of BPA, which may indicate a crosstalk between the non-genomic initiation effects mediated by PLC/PKC/IP3R signaling and genomic responses of BPA mediated by the estrogen receptor (ESR). In vitro exposure to a higher concentration of BPA caused cell damage and plasma membrane injury with increased LDH release and TAG content; both effects were dependent on intracellular Ca2+ and mediated by IP3R. Furthermore, BPA potentially induced liver damage, as demonstrated by increased GGT activity. In conclusion, in vitro effect of BPA in a low concentration triggers cytosolic Ca2+ overload and activates downstream protein kinases pointing to a crosstalk between its non-genomic and genomic effects of BPA mediated by ESR. Moreover, in vitro exposure to a higher concentration of BPA caused intracellular Ca2+-dependent testicular cell damage and plasma membrane injury. This acute toxicity was reinforced by increased testicular LDH release and GGT activity in the liver.

Acute exposure to bis(2-ethylhexyl)phthalate disrupts calcium homeostasis, energy metabolism and induces oxidative stress in the testis of Danio rerio.

Bis(2-ethylhexyl)phthalate (BEHP) negatively affects testicular functions in different animal species, disturbing reproductive physiology and male fertility. The present study investigated the in vitro acute effect of BEHP on the mechanism of action of ionic calcium (Ca2+) homeostasis and energy metabolism. In addition, the effect of BEHP on oxidative stress was studied in vitro and in vivo in the testis of Danio rerio (D. rerio). Testes were treated in vitro for 30 min with 1 µM BEHP for 45Ca2+ influx measurements. Testes were also incubated with 1 µM BEHP for 1 h (in vitro) or 12 h (in vivo) for the measurements of lactate content, 14C-deoxy-d-glucose uptake, lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT) activity, total reactive oxygen species (ROS) production and lipid peroxidation. In addition, the effect of BEHP (1 µM) on GGT, glutamic oxaloacetic transferase (GOT) and glutamic pyruvic transferase (GPT) activity in the liver was evaluated after in vivo treatment for 12 h. BEHP disturbs the Ca2+ balance in the testis when given acutely in vitro. BEHP stimulated Ca2+ influx occurs through L-type voltage-dependent Ca2+ channels (L-VDCC), transitory receptor potential vaniloid (TRPV1) channels, reverse-mode Na+/Ca2+ exchanger (NCX) activation and inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). BEHP affected energy metabolism in the testis by decreasing the lactate content and LDH activity. In vitro and in vivo acute effects of BEHP promoted oxidative stress by increasing ROS production, lipid peroxidation and GGT activity in the testis. Additionally, BEHP caused liver damage by increasing GPT activity.

Investigating the role of prostaglandin receptor isoform EP4b in zebrafish ovulation.

Prostaglandins (PGs) are a class of fatty acid-derived hormones that play an essential role in the regulation of ovulation of teleosts. This study investigated the various isoforms of ovarian PG receptors in the zebrafish ovary and their role in ovulation. Using real time qPCR, six PG receptor isoforms (ptger1a, ptger1b, ptger2a, ptger4a, ptger4b, and ptgfr) were shown to be expressed in the ovary. Only the PG receptor isoform ptger4b was upregulated at the time of ovulation in vivo, or following treatment in vivo with Ovaprim, which contains a gonadotropin releasing hormone analogue and a dopamine receptor antagonist and stimulates ovulation. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) in vitro also induced expression of EP4b mRNA. Females ovulate in vivo after injection with Ovaprim, or injection with Ovaprim and inhibitors of EP1 (ONO-8130) or EP2 (TG4-155) function; they do not ovulate when injected with Ovaprim and an EP4 inhibitor (GW237368x). These findings suggest that the EP4 receptor, in particular the EP4b isoform, is essential for ovulation.

Effects of atrazine on fish, amphibians, and reptiles: update of the analysis based on quantitative weight of evidence.

Quantitative weight of evidence (QWoE) provides a framework and process for evaluating different toxicological studies based on quality and relevance of the results. This framework allows for data from these studies to be combined in separate lines of evidence to address causality, and relevance to environmental risks. In 2014, such a QWoE that examined the body of available company reports and peer reviewed literature regarding the effects of the herbicide atrazine on fish, amphibians, and reptiles was published. Since that time, new studies have been conducted and/or published. One of the advantages of the QWoE framework is that additional information can be added as it becomes available. Thus, these new studies were evaluated in the same manner as previously and the new data incorporated into the existing QWoE. As before, the new updated QWoE was based on the same process of objective scoring of individual studies with respect to the quality of the methods and the relevance of individual responses to the apical endpoints of survival, growth, development, and reproduction. These new data did not identify new responses or indicate any relevant effects of atrazine. The new updated QWoE analysis concluded that atrazine does not adversely affect fish, amphibians, and reptiles, at environmentally relevant concentrations (<100 µg atrazine/L), which is consistent with the previous conclusions. These new studies and data are discussed in this paper and the accompanying supplement information provides detailed and transparent information to support these conclusions.

An International Perspective on the Tools and Concepts for Effluent Toxicity Assessments in the Context of Animal Alternatives: Reduction in Vertebrate Use.

Since the 1940s, effluent toxicity testing has been used to assess potential ecological impacts of effluents and help determine necessary treatment options for environmental protection prior to release. Strategic combinations of toxicity tests, analytical tools, and biological monitoring have been developed. Because the number of vertebrates utilized in effluent testing is thought to be much greater than that used for individual chemical testing, there is a new need to develop strategies to reduce the numbers of vertebrates (i.e., fish) used. This need will become more critical as developing nations begin to use vertebrates in toxicity tests to assess effluent quality. A workshop was held to 1) assess the state of science in effluent toxicity testing globally; 2) determine current practices of regulators, industry, private laboratories, and academia; and 3) explore alternatives to vertebrate (fish) testing options and the inclusion of modified/new methods and approaches in the regulatory environment. No single approach was identified, because of a range of factors including regulatory concerns, validity criteria, and wider acceptability of alternatives. However, a suite of strategies in a weight-of-evidence approach would provide the flexibility to meet the needs of the environment, regulators, and the regulated community; and this "toolbox" approach would also support reduced reliance on in vivo fish tests. The present Focus article provides a brief overview of wastewater regulation and effluent testing approaches. Alternative methodologies under development and some of the limitations and barriers to regulatory approaches that can be selected to suit individual country and regional requirements are described and discussed. Environ Toxicol Chem 2018;37:2745-2757. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Exposure to a Brazilian pulp mill effluent impacts the testis and liver in the zebrafish.

While many studies have shown that pulp mill effluents can affect ovarian physiology in fish, far fewer studies have considered the effects in males. We conducted a lab study to examine the effects of effluent from a Brazilian pulp and paper mill on hepatic and testicular morphology and various aspects of testicular physiology in the zebrafish Danio rerio. Males were exposed to lab water (control) or 4% effluent for 14 days. Effluent exposure did not affect testis size as measured by the gonadosomatic index, but contributed to morphological changes in the seminiferous tubules. The number of cysts with histopathological changes was elevated in effluent-exposed fish and the number of cysts containing spermatids was significantly reduced. The testis of effluent exposed fish had reduced levels of lactate, elevated lactate dehydrogenase activity, increased levels of reactive oxygen species and reduced levels of phosphorylated P38 mitogen-activated protein kinase (pP38 MAPK). Separate studies showed that the addition of lactate to testicular tissue incubated in vitro increased the activation of P38 MAPK. Effluent exposure also increased vacuolization, necrosis, apoptosis, hyperemia, and fat infiltration of the hepatocytes. Collectively, we provide evidence of short term effects of pulp mill effluent on testicular and hepatic physiology and biochemistry in the zebrafish.

Assessing recovery of in vitro steroid production in male rainbow darter (Etheostoma caeruleum) in response to municipal wastewater treatment plant infrastructure changes.

The present study examined in vitro 11-ketotestosterone and testosterone production by the testes of rainbow darter (Etheostoma caeruleum) collected from selected reference sites and downstream of 2 municipal wastewater treatment plants (MWWTPs; Waterloo and Kitchener) on the central Grand River (Ontario, Canada), over a 6-yr period (2011-2016). The main objective was to investigate if infrastructure upgrades at the Kitchener MWWTP in 2012 resulted in a recovery of this response in the post-upgrade period (2013-2016). Two supporting studies showed that the fall season is appropriate for measuring in vitro sex steroid production because it provides stable detection of steroid patterns, and that the sample handling practiced in the present study did not introduce a bias. Infrastructure upgrades of the Kitchener MWWTP resulted in significant reductions in ammonia and estrogenicity. After the upgrades, 11-ketotestosterone production by MWWTP-exposed fish increased in 2013 and it continued to recover throughout the study period of 2014 through 2016, returning to levels measured in reference fish. Testosterone production was less sensitive and it lacked consistency. The Waterloo MWWTP underwent some minor upgrades but the level of ammonia and estrogenicity remained variable over time. The production of 11-ketotestosterone and testosterone in rainbow darter below the Waterloo MWWTP was variable and without a clear recovery pattern over the course of the present study. The results of the present study demonstrated that measuring production of sex steroids (especially 11-ketotestosterone) over multiple years can be relevant for assessing responses in fish to environmental changes such as those resulting from major infrastructure upgrades. Environ Toxicol Chem 2018;37:501-514. © 2017 SETAC.