Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology

Published by Elsevier
Online ISSN: 1532-0456
Publications
Amount of proteins in ovaries (mgypaired ovary) from females of T. molitor when treated topically with 5 and 10 mg RH-0345 at day 0 and 2 after adult emergence
Article
RH-0345 belongs to a new group of insect growth regulators (IGRs) with a benzoylhydrazine structure that mimic the action of the natural insect molting hormone 20-hydroxyecdysone. After topical application on female adult beetles of mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), first oviposition was delayed, the number of eggs per female was reduced by 32%, the follicular epithelium was thinner (-33%) during sexual maturation, the size of deposited eggs was reduced, and egg viability was lost by 68%. Treatment with RH-0345 had also reduced the ovarian protein content and two protein bands were missing in the ovaries. Ultrastructural observations of the ovaries at the end of vitellogenesis in treated females, however, showed no evident differences with the fine structure of both follicular cells and oocytes in controls. In addition, we measured the amount of ecdysteroids in the medium of treated ovary cultures in vitro and in the eggs deposited by treated females. Possible action sites with the reproductive system at different levels in T. molitor are discussed for this novel group of IGRs.
 
Article
Polychlorinated naphthalenes are environmentally relevant compounds that are measured in biota at concentrations in the μg/kg lipid range. Despite their widespread occurrence, literature data on the accumulation and effects of these compounds in aquatic ecosystems are sparsely available. The goal of this study was to gain insights into the biomagnification and effects of 1,2,3,5,7-pentachloronaphthalene (PeCN52) in an experimental food chain consisting of benthic worms and juvenile rainbow trout. Worms were contaminated with PeCN52 by passive dosing from polydimethylsiloxane silicone. The contaminated worms were then used to feed the juvenile rainbow trout at 0.12, 0.25 or 0.50μg/g fish wet weight/day, and the resulting internal whole-body concentrations of the individual fish were linked to biological responses. A possible involvement of the cellular detoxification system was explored by measuring PeCN52-induced expression of the phase I biotransformation enzyme gene cyp1a1 and the ABC transporter gene abcb1a. At the end of the 28-day study, biomagnification factors were similar for all dietary intake levels with values between 0.5 and 0.7kg lipidfish/kg lipidworm. The average uptake efficiency of 60% indicated that a high amount of PeCN52 was transferred from the worms to the fish. Internal concentrations of up to 175mg/kg fish lipid in the highest treatment level did not result in effects on mortality, behavior, or growth of the juvenile trout, but were associated with induction of phase I metabolism which was evident from the significant up-regulation of cyp1a1 expression in the liver. In contrast, no changes were seen in abcb1a transcript levels. Copyright © 2015. Published by Elsevier Inc.
 
Article
The antiprotozoal activity of newly synthesised compounds, all [1,2,4]triazolo [1,5a]pyrimidine derivatives, was tested against the protozoan parasites Trypanosoma cruzi, Leishmania donovani and Phytotmonas staheli. Six of these compounds significantly inhibited in vitro cell growth of the epimastigote forms of T. cruzi, and the promastigote forms of L. donovani and P. staheli. Some of the compounds reached complete growth inhibition at 1 microg/ml for 48 h of parasite/drug interaction. None of the compounds tested showed significant toxicity against cells of Aedes albopictus, mouse macrophages J-774A.1 and Lycopersicum esculentum at dosages five times greater than used against parasites.
 
Article
The in vivo metabolic fate of 1,8-cineole was investigated in six male koalas. Koalas were fed ad lib a diet of Eucalyptus cephalocarpa leaf with a 1,8-cineole concentration of 2.53+/-0.70% dry mass of leaf, corresponding to a 1,8-cineole intake of 2.4+/-1.1 mmol/kg (3.1+/-1.3 g). Urine and faeces were collected for 24 h and metabolites identified by GC-MS and LC-MS. Metabolites were quantified before and after hydrolysis with beta-glucuronidase to give free and total levels, respectively. Fractional recovery of ingested 1,8-cineole was 1.3+/-0.4 and 1.4+/-0.4 (mean+/-S.D.) for free and total measurements, respectively. Seven metabolites were identified and quantified: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. The hydroxycineolic acids dominated the metabolite profile (85%). 7,9-Dicineolic acid, a novel metabolite of 1,8-cineole, accounted for almost 10% of the recovered dose making it the second most abundant metabolite after 7-hydroxy-9-cineolic acid (77%). Together, the less oxidised metabolites, the hydroxycineoles and cineolic acid, accounted for only 5% of the cineole consumed. Significant conjugation only occurred with four minor, less oxidised, alcohol and carboxylic acid metabolites. We have shown that the koala detoxifies and eliminates 1,8-cineole primarily by extensive oxidation without utilising conjugation pathways.
 
Article
Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.
 
Article
A number of compounds were isolated from the medicinal plant Aster tataricus including shionone, epifriedelinol, quercetin, kaempferol, scopoletin, emodin, aurantiamide acetate and 1,7-dihydroxy-6-methyl-anthraquinone. The compounds were compared with regard to their ability in inhibiting hemolysis of rat erythrocytes induced by 2'-2' azobis (2-amidinopropane) dihydrochloride, lipid peroxidation using the FeSO(4)-ascorbic acid system, and generation of superoxide radicals using a phenazine methosulfate-nicotinamide adenine dinucleotide system. The effects on the Fe-bleomycin-induced DNA damage reflected pro-oxidant activity. Quercetin and kaempferol were most potent in inhibiting hemolysis, lipid peroxidation and superoxide radical generation. Scopoletin and emodin were similar to quercetin and kaempferol in inhibiting superoxide radical generation and second to them in inhibiting lipid peroxidation. Aurantiamide acetate exhibited some inhibitory activity toward superoxide radical generation. 1,7-dihydroxy-6-methyl-anthraquinone exerted an inhibitory activity only on superoxide radical generation. Shionone and epifriedelinol did not display any antioxidant activity. Quercetin and kaempferol, but not the remaining compounds, exhibited some pro-oxidant activity.
 
Article
The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone, progesterone and cortisol was analysed in common carp (Cyprinus carpio L.). Carp were injected intraperitoneally (1 and 5 microg/kg fish) with each steroid and the activity of kidney macrophages was recorded 1 and 3 days post-injection. Bovine serum albumin-injected fish served as controls. Phagocytosis, superoxide anion and nitric oxide production were suppressed in all sex steroid treatments, and the suppression was in a dose-dependent manner. Cortisol treatment also inhibited the immune parameters to an extent similar to each of the steroid hormone. These results show that sex steroids can modulate the function of the phagocytic cells.
 
Article
Juvenile rainbow trout (Oncorhynchus mykiss) were subjected to a 2-day radioactive pulse of 110mAg at 11.9 microg/l (as AgNO3), followed by a 19-day post-tracer exposure to non-radioactive Ag(I) (3.8 microg/l). The distribution of 110mAg in the gills, liver, intestine, kidney, brain and remaining carcass was investigated over a 19-day post-tracer period. Initially, the intestine contained the highest proportion of the 110mAg burden (34%), however, by day 8, less than 5% of the total radioactivity remained in this tissue. The majority of the 110mAg eliminated from the intestine appeared to distribute to the liver. Eventually, the 110mAg content in the liver accounted for as much as 65% of the total radioactivity in the fish. Apart from the liver and intestine, only the gills and carcass contained any appreciable amount (>5%) of the total body 110mAg content. Liver and gill samples were fractionated using differential centrifugation techniques to discern the subcellular distribution of 110mAg in these tissues. In the liver, the 110mAg levels in the cytosolic fraction increased from 35% to 72% of the total cellular burden between days 8 and 19, respectively. The radioactive pulse in the gills was predominantly found in a membrane compartment termed the nuclear fraction ( approximately 60% of the total). Little change was observed over time (day 8 to day 19) to the subcellular distribution of Ag in the gills. Using size-exclusion chromatography, most ( approximately 70%) of the 110mAg content in the liver cytosol eluted at a molecular weight characteristic of metallothionein. The cytosolic distribution of 110mAg in gills was quite diffuse, occurring primarily in the heavy molecular weight fractions.
 
Article
We investigated maternal and fetal tissue distribution of DW-116, a newly developed fluoroquinolone with a broad antibacterial spectrum against both G(+) and G(-) bacteria, in pregnant rats. After oral administration of [14C]-DW-116 (labeled 1 mg and unlabeled 500 mg/kg) to female rats on the 18th day of gestational, groups of three rats were killed at various time points up to 24 h, and plasma and tissues were collected, processed and analyzed. [14C]-DW-116 was rapidly absorbed, and distributed into the maternal and fetal tissues, and it declined in a biphasic manner with elimination half-lives (t(1/2)) of 10-15 h and mean residence times (MRT(0-24 h)) of 4-9 h. The radioactivity in most tissues of both dams and fetus reached its peak within 1 h and radioactivity levels of up to 10-25% of the peak level were maintained until 24 h after dosing. Among various tissues, the radioactivity in the maternal lungs was the highest (27 times that of plasma) at the C(max). Radioactivity in other tissues including liver, kidney, heart, lung, brain, spleen, mammary gland, placenta, ovary and uterus was higher than that in the maternal plasma (one- to three-fold). The tissue-to-plasma partition coefficient (K(p), AUC(0-24 h,tissue)/AUC(0-24 h,plasma)) of [14C]-DW-116 in maternal tissues was highest in the lung (K(p)=3.7), followed by the spleen (2.2), kidney (2.0), liver (1.8), heart (1.5), placenta (1.3), brain (1.3), ovary (1.1), uterus (1.1), and mammary gland (1.0). The tissue-to-plasma partition coefficient values in fetal tissues were heart (K(p)=2.2), kidney (2.1), liver (1.9), lung (1.6) and brain (1.4). When lactating rats were given a single oral dose of [14C]-DW-116, the radioactivity was rapidly secreted into the milk with K(p) of 1.7 at T(max) (0.5 h). These results indicate that DW-116 or its related metabolite(s) rapidly cross the blood-placenta and blood-milk barrier, extensively distribute into the fetal tissues, and are eliminated from the body in a prolonged manner. This study sheds insights into the maternal and fetal tissue distribution of DW-116 and will be useful for assessing both therapeutic and toxicological relevance of DW-116 in pregnant subjects.
 
Article
The 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) interconvert 11beta-hydroxysteroids such as cortisol into 11-oxosteroids such as cortisone. In most mammals, 11beta-HSD 1 is expressed predominantly in the liver and is active in both the oxidative (cortisol to cortisone) and dehydrogenase (cortisone to cortisol) directions, whilst 11beta-HSD 2 is expressed predominantly in the kidney and functions as a pure oxidative enzyme. We have investigated 11beta-HSD 1 activity in the Australian koala (Phascolarctos cinereus) and have found no activity (either reductive or oxidative) in hepatic microsomes. Immunoblot analysis of koala hepatic microsomes, using an 11beta-HSD 1 antibody raised against the mouse enzyme, failed to identify immunoreactive protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) of koala liver mRNA and genomic PCR using primers designed against highly conserved regions of 11beta-HSD 1 nucleotide sequences were also negative. Furthermore, Southern and Northern blot analysis of koala genomic DNA and mRNA, respectively, confirmed that the koala lacks the 11beta-HSD 1 gene and gene transcript. These results support the fact that the lack of hepatic 11beta-HSD 1 activity in the koala is due to the absence of the 11beta-HSD 1 gene, and this absence is novel among mammalian species studied to date.
 
Article
Temperature is known to influence xenobiotic retention in fish. The effect of acute and acclimatory temperature change upon Rhodamine 123 (Rho123) permeability through an in vitro catfish multi-segment (3) everted sac intestinal wall model was examined in a 9 cell matrix of acclimation and assay temperatures (10, 20 and 30 degrees C). Changes in Rho123 permeability were examined in context with membrane fluidity, xenobiotic solubility and intestinal morphology. When assayed at the acclimation temperature greater Rho123 permeability was noted at warmer acclimation temperatures for the proximal and middle intestinal segments, while the distal segment exhibited little change and apparent compensation across temperatures. Rho123 permeability was increased as assay temperatures were elevated above the acclimation temperature for most comparisons. Cold acclimation significantly increased total intestinal length (43.2%) and proximal intestine weights while total body weights did not differ. Brush border membranes (BBM) increased fluidity with increased assay temperatures, however, composite anisotropy lines were not significantly different between acclimation treatments. In an additive manner, the membrane probe DPH exhibited increased solubility in BBM with increases in acclimation and assay temperatures. Compositely, these results suggest that acclimation and acute temperature change may differentially influence xenobiotic permeability among intestinal segments with interacting mechanisms.
 
Article
The objective of this study was to investigate the impact of short-term exposure to polychlorinated biphenyls on the acute stress response in rainbow trout. Fish were exposed to dietary Aroclor1254 (10mg kg(-1) body mass/day) for 3 days and then subjected to a 3-min handling disturbance and sampled over a 24h recovery after the stressor exposure. In the pre-stress fish, PCB exposure significantly elevated aryl hydrocarbon receptor (AhR) and cytochrome P4501A1 (Cyp1A1) mRNA abundance and Cyp1A protein expression confirming AhR activation. There was no significant effect of PCB on plasma cortisol and glucose levels, while plasma lactate levels were significantly elevated compared to the sham group. PCB exposure significantly elevated liver glycogen content and hexokinase activity, whereas lactate dehydrogenase activity was depressed. Short-term PCB exposure did not modify the acute stressor-induced plasma cortisol, glucose and lactate responses. Liver glycogen content dropped significantly after stressor exposure in the PCB group but not in the sham group. This was matched by a significantly higher liver LDH activity and a lower HK activity during recovery in the PCB group suggesting enhanced glycolytic capacity to fuel hepatic metabolism. Liver AhR, but not Cyp1A1, transcript levels were significantly reduced during recovery from handling stressor in the Aroclor fed fish. Collectively, this study demonstrates that short-term PCB exposure may impair the liver metabolic performance that is critical to cope with the enhanced energy demand associated with additional stressor exposure in rainbow trout.
 
Effects of exposure to Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) on circulating levels of T 4 and T 3 in Atlantic croaker. Bars represent average TH 
Effects of exposure to Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) on circulating levels of T 4 and T 3 in female Atlantic croaker. Bars represent 
Total PCB accumulation in the livers of croaker exposed to the high dose of Aroclor 1254 (1.0 mg/kg body mass/day) showed a positive correlation with the hepato-somatic indices (HSIs) of individual fish. 
Effects of exposure to PCB 153 (0.1 and 1.0 mg/kg body mass/day) on circulating levels of T 4 and T 3 in Atlantic croaker. Bars represent average TH 
Effects of exposure to PCB 77 on circulating levels of T 4 and T 3 in 
Article
Atlantic croaker (Micropogonias undulatus) were exposed to the polychlorinated biphenyl (PCB) mixture (Aroclor 1254) or one of three individual congeners (planar PCB 77 or ortho-substituted PCB 47 and PCB 153) in the diet for 30 days to investigate the effects of PCBs on circulating thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) decreased plasma T3 levels consistently, but the effects on T4 levels were inconsistent from year to year. Exposure to PCB 153 (0.1 and 1.0 mg/kg body mass/day) significantly lowered both T4 and T3, while PCB 47 at the same doses had no effect on thyroid hormone levels. The lower doses of PCB 77 (0.004, 0.01 and 0.02 mg/kg body mass/day) had no effect on T4 or T3, whereas the highest dose (0.1 mg/kg body mass/day) increased T4 levels significantly. The results of the present study demonstrate that exposure to PCBs at environmentally realistic concentrations can have profound effects on the thyroid status of Atlantic croaker. The ortho-substituted PCB 153 appears to contribute at least partially to the deleterious effects of Aroclor 1254 on thyroid status, whereas the planar PCB 77 at concentrations present in the mixture is unlikely to alter thyroid hormone levels.
 
Article
Glutathione peroxidases (GPx) and glutathione S-transferases (GST) are essential enzymes of the cellular defense system. The aim of this work was the identification of GPx transcript in a freshwater bivalve, Unio tumidus, and the effects of Aroclor 1254 on GPx and pi-class GST (pi-GST) expression pattern. The GPx full-length coding sequence was obtained by reverse transcription PCR using degenerated primers followed by 5' and 3' rapid amplification of cDNA ends. The GPx cDNA encodes a protein of 232 amino acids. The 72nd amino acid corresponds to a selenocysteine encoded by a TGA codon. Residues essential to the enzymatic function are conserved in GPx of U. tumidus. Specific amplifications of the Se-GPx mRNA from U. tumidus were performed on the digestive gland, the excretory system and the gills. Se-GPx expression level is highest in the digestive gland. No induction of the Se-GPx was observed at the transcriptional level in the digestive gland and the excretory system of Aroclor-treated mussels, while an increase of the pi-GST mRNA level was observed in the excretory system.
 
Article
We evaluated the effects of two organochlorinated environmental contaminants, Endosulfan and Aroclor 1254 on peripheral thyroid hormone metabolism and thyroid hormone plasma levels in Nile tilapia (Oreochromis niloticus). Tilapia were exposed through diet to 0.1 and 0.5 microg g(-1) of Endosulfan and 0.5 microg g(-1) of Aroclor 1254 for 21 and 35 days. Decreased plasma T4 and rT3 levels were observed in tilapia exposed to the lower dose of Endosulfan, while treatment with a higher dose and Aroclor 1254 produced no changes. Plasma T3 levels were not affected by these compounds. Hepatic type I deiodinase (D1) activity was depressed by a lower dose of Endosulfan and hepatic type III (D3) activity was increased following 35 days of exposure to the lower dose of Endosulfan and following 21 and 35 days of exposure to Aroclor 1254; while type II (D2) remained unchanged in liver as well as in all other organs analysed. Apart from hepatic D3 activity, Endosulfan and Aroclor 1254 also increased D3 activity in gill, but not in other tested organs. It is concluded that dietary exposure of tilapia to Endosulfan or Aroclor 1254 can lead to changes in circulating thyroid hormone levels and/or in peripheral thyroid hormone metabolism. The changes in hormone metabolism differ between tissues, eventually reflecting tissue-specific differences in adaptation.
 
Article
Glutathione S-transferases (GSTs) are a family of multifunctional enzymes involved in cellular detoxification that catalyze the attachment of electrophilic substrates to glutathione. Two classes of GSTs related to the rho and sigma classes of enzymes in Antarctic bivalves have been cloned from Laternula elliptica. The full-length cDNA of rho class GST (leGSTr) is 1530bp in length and contains an open reading frame (ORF) of 672bp encoding 223 amino acid residues. The deduced amino acid sequences of this gene have 41% and 40% identity to rho class GSTs from Ctenopharyngodon idella and Pleuronectes platessa, respectively. The sigma class GST (leGSTs) cDNA, however, is 1127bp in length and contains an ORF of 696bp encoding 231 amino acid residues. The deduced amino acid sequences share only 22% identity with sigma class GST from Xenopus laevis. The transcriptional expression of leGSTr, leGSTs, and leGSTp cloned in our previous study were examined using real-time polymerase chain reaction in response to exposure to a polychlorinated biphenyl (PCB) mixture. The expressions of these three GST transcripts were rapidly upregulated, although they showed different expression levels and patterns within each isoform. Moreover, leGSTs was the most upregulated in the gill and digestive gland in response to PCB exposure. The recombinant GSTs were highly expressed in transformed Escherichia coli, and their kinetic properties were studied with various substrates. As a result, the three classes of GSTs were found to have diverse biological functions and were responsible for different enzymatic features.
 
Article
Integrated effects of polychlorinated biphenyl (PCB) and nutritional status on responses to handling disturbance were investigated in the Arctic charr (Salvelinus alpinus). The fish were orally contaminated with Aroclor 1254 and held either with or without food for 5 months before they were subjected to a 10-min handling disturbance. Food-deprived fish were given 0, 1, 10 or 100 mg PCB kg(-1) and the fed fish 0 or 100 mg PCB kg(-1). Plasma cortisol, glucose and lactate levels were measured at 0 (pre-handling), 1, 3, 6 and 23 h after the handling disturbance. Food-deprived control fish had elevated plasma cortisol levels compared with fed fish before handling. These basal cortisol levels were suppressed by PCB in food-deprived fish, and elevated by PCB in fed fish. The immediate cortisol and glucose responses to handling disturbance were suppressed by PCB in a dose-dependent way in food-deprived fish. Although these responses were also lowered by PCB in the fed fish, the effect was much less pronounced than in food-deprived fish. There were only minor effects on plasma lactate responses. Our findings suggest that the stress responses of the Arctic charr are compromised by PCB and that the long-term fasting, typical of high-latitude fish, makes these species particularly sensitive to organochlorines such as PCB.
 
Article
Polychlorinated biphenyls (PCB) and other aryl hydrocarbon receptor (AHR) agonists induce oxidative stress and alter membrane lipid peroxidation and fluidity. This study tested the hypothesis that PCB-induced changes in membrane properties impact membrane beta-adrenoceptor (beta-AR) affinity and capacity in chick embryo hepatocytes. Embryos were injected into the air cell with 1.6 microg 3,3',4,4',5-pentachlorobiphenyl (PCB 126)/kg egg at day 0, and incubated to day 19 when livers were removed. This dose resulted in hepatic PCB 126 levels of 0.67 ng/g liver or 10.2 ng/g liver lipid; levels in untreated embryos were non-detectable. Hepatic microsomal EROD activity was elevated by approximately 12-fold and embryo mortality was significantly increased compared with the untreated group. Hepatic lipid peroxidation increased and membrane order (steady-state fluorescence anisotropy values) decreased with in ovo PCB 126 exposure. Consistent with changes in membrane structure, hepatic beta-AR affinity for CGP 12177 significantly decreased (Kd increased) without changes in receptor numbers. This study demonstrates that in ovo exposure to PCB 126 in chick eggs significantly impacted embryo survival, and this was correlated with altered hepatic membrane structure and ultimately membrane function.
 
Article
Treatment of chickens as pre-incubation embryos with TCDD or PCB-126 altered fatty acid concentrations in their plasma 21 days later, compared with their oil vehicles (sunflower and corn oils, respectively). TCDD increased the concentrations of total fatty acids, lipid classes (phospholipids and cholesterol ester), fatty acid families (saturated, n-7 and n-6), and many specific fatty acids. The only fatty acid concentrations decreased by TCDD treatment were those of cholesterol ester fatty acids 20:3n3 and 24:6n3 and overall plasma 24:6n3. In contrast, PCB-126 treatment decreased total phospholipid, saturated and plasmogen fatty acid concentrations with generally decreasing trends in specific fatty acid concentrations. However, both TCDD and PCB-126 treatments increased total 22:1n9 and decreased 24:6n3 concentrations compared with their respective vehicles. The potential relationship between those fatty acid concentrations altered by toxicant treatment and alterations in brain symmetry was then examined using correlation analysis. Several fatty acid concentrations were significantly correlated with differences in brain morphology between the right and left hemispheres and these potential associations were different between toxicant and vehicle.
 
Article
Glutathione transferases (GSTs) are phase II enzymes that detoxify a wide range of toxicants and reactive intermediates. One such class of toxicants is the ubiquitous polycyclic aromatic hydrocarbons (PAHs). Certain PAHs are known to cause developmental cardiac toxicity in fish. Herein, we explored the role of GST pi class 2 (GSTp2) in PAH- and PCB-induced cardiac toxicity in zebrafish (Danio rerio) embryos. We measured expression of GSTp2 in embryos exposed to individual and co-exposures of the PAHs benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), and fluoranthene (FL) as well as 3,3',4,4',5-pentachlorobiphenyl (PCB-126). GSTp2 mRNA expression was induced by exposure to BkF, BaP, PCB-126, and BaP+FL and BkF+FL co-exposure. A splice junction morpholino was then used to knockdown GSTp2 in developing zebrafish. GSTp2 knockdown exacerbated the toxicity caused by co-exposures to BkF+FL and BaP+FL. However, GSTp2 knockdown did not affect PCB-126 toxicity. These results further suggest that pi class GSTs serve a protective function against the synergistic toxicity caused by PAHs in developing zebrafish.
 
Article
Little is known about the sensitivity of teleost post-embryonic developmental stages (larval and metamorphic) to dioxin-like compounds. Larval and metamorphosing summer flounder (Paralichthys dentatus) were exposed to the dioxin-like polychlorinated biphenyl congener PCB 126, to compare their sensitivity to other fish species early life stages, and to document effects on metamorphic development, including degree of eye migration and gastric maturation. Median lethal doses (LD 50s) ranged between 30 and 220 ng/g wet mass, indicating that pre- and early-metamorphic stages of summer flounder are equally sensitive to the embryos of some of the most vulnerable fish species tested. Consistent with the presence of a functional aryl hydrocarbon receptor pathway, dose-dependent induction of cytochrome P-4501A (CYP1A) at four days post-exposure was observed in liver, stomach, intestine, and kidney of metamorphosing larvae. Stage-dependent differences in the epithelial distribution of CYP1A immunoreactivity were observed in the developing stomach of fish exposed to relatively high PCB 126 doses. A single sublethal dose (15 ng/g) delayed metamorphic progress (determined by the degree of eye migration), and resulted in abnormally high levels of cell proliferation and abnormal gastric gland morphology in late metamorphic stages. These results suggest that the post-embryonic larval and metamorphic stages of summer flounder, and potentially other fish species with complex life histories, are vulnerable to the effects of dioxin-like compounds, including lethality, developmental delay, and malformations.
 
T4ORD (A), T4IRD (B) and rT3ORD (C) activities in tissues of American plaice measured in vitro at a substrate concentration of 0.5 nM. Each bar represents the mean ( 9 S.E.M.) from seven different plaice each measured in triplicate. 
The effect of incubation temperature on deiodination rates in plaice liver and brain. Each point represents the mean from triplicate measures from a single pool of microsomes. 
The effect of incubation temperature on deiodination rates in plaice liver and brain. Each point represents the mean from triplicate measures from a single pool of microsomes. Fig. 5. Liver T4ORD (top) and T4IRD (bottom) activities in plaice injected with 0 (n = 8), 5 (n = 12 for T4ORD, n = 11 for T4IRD) or 25 ng (n =12) PCB 77 per g bm and sampled at 1 week, and 0 (n =10), 5 (n = 10) or 25 ng (n = 10) PCB 77 per g bm after 4 weeks. Differences (P 50.05) among means at any one sampling time are indicated by different superscripts. 
Article
We have described the tissue distribution and properties of thyroid hormone (TH) deiodination activities of the marine American plaice, Hippoglossoides platessoides. We then studied the 1- or 4-week responses of the plaice liver and brain deiodination activities and the plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels to an intraperitoneal injection (5-500 ng/g) of the polychlorinated biphenyl (PCB) congeners 77 (3,3'-4,4'-tetrachlorobiphenyl) or 126 (3,3',4,4',5-pentachlorobiphenyl). T4 and 3,3'5'-triiodothyronine (rT3) outer-ring deiodination (ORD) activities were greater in liver than in kidney, gill, heart, brain, intestine or muscle; inner-ring deiodination (IRD) activity occurred in all tissues but was consistently higher in brain. Deiodination characteristics (optimal pH, optimal dithiothreitol concentration, responses to inhibitors and apparent Km values of 0.6-4 nM) fell in the same rage as those of low-Km deiodinases in other teleosts. Deiodination activities were maximal when assayed at 25 degrees C but uniformly low over the natural range of 0-9 degrees C. Neither PCB 77 nor PCB 126 altered brain T4ORD activity or plasma T4 levels (P < 0.05). However, at 1 week post injection hepatic T4ORD activity was increased and plasma T3 levels lowered by PCB 77 (5 and 25 ng/g), while hepatic IRD activity was increased by PCB 126 (50 and 500 ng/g). Neither PCB 77, PCB 126 nor selected hydroxylated. PCBs given in vitro compared with T4 for binding sites on plasma proteins or altered hepatic deiodination activity, indicating no direct action on plasma proteins or deiodinases We conclude that plaice TH deiodination tissue distribution and characteristics resemble those of other teleosts. Deiodination activities are low at natural assay temperatures but at 1 week show some responses to PCBs 77 and 126.
 
Article
Differential gene expression profiling was performed with a cDNA microarray in the liver tissue of the medaka fish, Oryzias latipes, after exposure to Arochlor 1260, a polychlorinated biphenyl (PCB) mixture, which is used as a coolant and insulating fluid for transformers and capacitors and is classified as a persistent organic pollutant. Twenty-six differentially expressed candidate genes were identified. The expression of 12 genes was up-regulated and that of 14 genes was down-regulated. These genes are associated with the cytoskeleton, development, endocrine/reproduction, immunity, metabolism, nucleic acid/protein binding, and signal transduction, or are uncategorized. The transcription of molecular biomarkers known to be involved in endocrine disruption (e.g., vitellogenins, choriogenins, and estrogen receptor alpha) was highly up-regulated. The same tendencies in gene expression changes were observed with real-time quantitative PCR (qRT-PCR) analysis, which was conducted to examine 12 selected candidate genes. These genes could be used as molecular biomarkers for biological responses to toxic chemicals, especially endocrine disrupting and carcinogenic chemical contamination in aquatic environments.
 
Article
Cytochrome P450 CYP2 family enzymes are important in a variety of physiological and toxicological processes. CYP2 genes are highly diverse and orthologous relationships remain clouded among CYP2s in different taxa. Sequence and expression analyses of CYP2 genes in diapsids including birds and reptiles may improve understanding of this CYP family. We sought CYP2 genes in a liver cDNA library of the common cormorant (Phalacrocorax carbo), and in the genomes of other diapsids, chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and anole lizard (Anolis carolinensis), for phylogenetic and/or syntenic analyses. Screening of the cDNA library yielded four CYP2 cDNA clones that were phylogenetically classified as CYP2C45, CYP2J25, CYP2AC1, and CYP2AF1. There are numerous newly identified diapsid CYP2 genes that include genes related to the human CYP2Cs, CYP2D6, CYP2G2P, CYP2J2, CYP2R1, CYP2U1, CYP2W1, CYP2AB1P, and CYP2AC1P. Syntenic relationships show that avian CYP2Hs are orthologous to CYP2C62P in humans, CYP2C23 in rats, and Cyp2c44 in mice, and suggest that avian CYP2Hs, along with human CYP2C62P and mouse Cyp2c44, could be renamed as CYP2C23, based upon the nomenclature rules. Analysis of sequence and synteny identifies cormorant and finch CYPs that are apparent orthologs of phenobarbital-inducible chicken CYP2C45. Transcripts of all four cormorant CYP2 genes were detected in the liver of birds from Lake Biwa, Japan. The transcript levels bore no significant relationship to levels of chlorinated organic pollutants in the liver, including polychlorinated biphenyls and dichlorodiphenyltrichloroethane and its metabolites. In contrast, concentrations of perfluorooctane sulfonate and perfluorononanoic acid were negatively correlated with levels of CYP2C45 and/or CYP2J25, suggesting down-regulation of expression by these environmental pollutants. This study expands our view of the phylogeny and evolution of CYP2s, and provides evolutionary insight into the chemical regulation of CYP2 gene expression in diapsids including birds.
 
Article
Muscarinic acetylcholine receptors (mAChRs) play a role in learning, memory and behavior in vertebrate animals. We measured the muscarinic cholinergic receptor levels in extracts from zebrafish (Danio rerio) brain by radioligand binding techniques. Saturation binding experiments with the radioligand [3H]-quinuclidinyl benzilate (QNB) were used to determine receptor number and relative affinity for several agonists and antagonists. Affinity at zebrafish brain receptors was relatively high with a K(d) of 40 +/- 5 pM. The number of receptors, represented by Bmax, was 63 +/- 16 fmol/mg protein. Oxotremorine and carbachol, agonists at muscarinic acetylcholine receptors, bound with displacement curves indicating multiple binding sites. In addition, oxotremorine bound with a higher affinity than did carbachol. The antagonist potency profile at zebrafish receptors in brain was determined to be atropine>pirenzipine>p-fluoro-hexahydro-sila-difenidol>otenzepad. The results obtained with zebrafish brain compare favorably to those found in insect, fish and mammalian species. Taken together, the binding results and favorable comparisons to mammalian systems indicate that zebrafish may provide a useful model organism for evaluating the role of cholinergic systems in learning, memory and behavior.
 
Article
Thioredoxin (abbreviated as Trx) is an important ubiquitous disulfide reductase, which can protect organisms against various oxidative stresses. In the present study, thioredoxin 1 (named as VpTrx1) and thioredoxin-related protein (named as VpTrp14) were identified from Venerupis philippinarum, respectively. Similar to most Trx1s, VpTrx1 possessed all conserved features critical for the fundamental structure and function of Trx1s, such as the conserved catalytic residues (C-G-P-C), but lacked the other cysteine residues, while VpTrp14 contained the conserved motif (C-P-D-C). Quantitative Real-time PCR assay showed that VpTrx1 and VpTrp14 transcripts were distributed in a wide array of tissues most abundantly expressed in the hepatopancreas. The expression of VpTrp14 mRNA in the hepatopancreas was significantly up-regulated after exposure to 10 and 40μg/L Cd, while the VpTrx1 expression level was kept relatively constant. Both the expression levels of VpTrx1 and VpTrp14 in the hepatopancreas were induced after exposure to Cu, and increased to the peak value at 96h under the 40μg/L Cu exposure. These results showed that VpTrp14 transcripts responded to metal stress more acutely than VpTrx1, and both Trxs responded to Cu stress more sensitively than Cd. Together, it was suggested that VpTrx1 and VpTrp14 perhaps played important roles in the antioxidant responses against metal stress in V. philippinarum.
 
Article
The present study examines the influence of Ca2+ as (CaSO4), dissolved organic carbon (DOC) and pH on chronic water-borne lead (Pb) toxicity to the larval fathead minnow (Pimephales promelas) under flow-through conditions. The 30 day LC50 for low hardness basic test water (19 mg CaCO3 L(-1)) was 39 (range: 27-51) microg dissolved Pb L(-1) and was greatly increased by increasing concentrations of CaSO4 and DOC to as much as 1903 (range: 1812-1992) mug dissolved Pb L(-1). Both reduced and increased pH (6.7 and 8.1, respectively) compared to control pH of 7.4 appeared to increase Pb toxicity substantially. Whole body Pb accumulation did not reflect water chemistry and thus exhibited no correlation with Pb induced mortality. One possible explanation for this lack of correlation is that mortality occurred predominantly during the first 4-6 days of exposure, whereas Pb accumulation was determined in surviving fish at the end of 30 days of exposure. Chronic Pb exposure resulted in a general iono-regulatory disturbance affecting K+, Na+ and Ca2+ homeostasis. However, recovery of Na+ and K+ levels and reversal of effects on Ca2+ homeostasis during continued exposure strongly suggest fathead minnow can acclimate to Pb. The gills accumulate the highest Pb concentrations during chronic exposure but the skeleton contains the largest mass of Pb by contributing up to approximately 80% of whole body Pb. In conclusion, water chemistry characteristics like Ca2+ and DOC should be considered for chronic water quality criteria.
 
Article
Interference between heavy metals and growth hormone (GH) on cell signaling has been previously demonstrated in fish cells. This study was aimed at assessing their effects on expression of the metallothionein isoforms MT-A and MT-B. The results indicate that all heavy metals induce MT-A more markedly than MT-B, but differences appeared when metals were combined with GH. For MT-B induction, a positive interference between metals and GH was observed for Zn(2+)/GH and Cd(2+)/GH, a negative interference for Hg(2+)/GH. With regards to MT-A, no interference was observed for Zn(2+)/GH and Hg(2+)/GH, while a negative interference occurred with Cu(2+)/GH and a positive interference with Cd(2+)/GH. The possible mechanisms underlying the differential regulation of metallothioneins include different signaling pathways. The results show that STAT5 and ERKs responded differently to different combinations, and Zn(2+)/GH and Cd(2+)/GH exerted a slight positive interference on ERK activation. On the other hand, a synergic rise in [Ca(2+)](i) occurred for all combinations except for Cu(2+)/GH. Our data suggest that the cross-talk between heavy metals and GH resulting in MT transcription modulation does not strictly depend on Ca(2+) signalling; (ii)ERK activation may represent the point of cross-talk between Zn(2+) or Cd(2+) and GH, converging on MT-B transcription, probably through a differential recruitment of transcription factors.
 
Article
The distribution and excretion of arsenobetaine in fish were investigated using whole body autoradiography and liquid scintillation counting. A single dose of synthesised [(14)C]arsenobetaine was orally administered to Atlantic salmon, Salmo salar L., and Atlantic cod, Gadus morhua L. Arsenobetaine was distributed to most organs within both species. Nevertheless, there were species differences in tissue distribution and excretory pattern. The highest level of arsenobetaine in Atlantic salmon was present in muscle tissue, while high levels of arsenobetaine were found in both muscle and liver (including gall bladder) from Atlantic cod. The results suggest that the major route of excretion was via urine, which seemed to be more important in Atlantic cod than in Atlantic salmon. Elimination of arsenobetaine via bile appeared to be negligible in both species.
 
Article
We examined the estrogenic effect of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on the rockfish, Sebastes schlegeli. We measured levels of plasma estradiol-17beta (E(2)) and testosterone (T) by radioimmunoassay (RIA). Plasma concentrations of T and 17alpha-hydroxyprogesterone in female and male fish injected with PCB 153 using two dosages (0.16 mg/kg body weight. and 0.57 mg/kg) did not differ significantly between sexes or from sham-injected controls of the same sex. Plasma concentrations of E(2) in females injected with PCB 153 (both levels) increased at 12 and 24 h. Concentrations of plasma vitellogenin (VTG) in females increased 72 h after injection with PCB 153 and reached 0.38 and 1.25 mg/mL, respectively. No VTG was detected in males injected with the same dosage. These results suggest that PCB 153 may lead to the production VTG in female rockfish through a synergistic effect with E(2), resulting in indirect disruption of the aromatization process.
 
Article
This study was undertaken to determine whether gulf toadfish (Opsanus beta) could metabolize ammonia from their environment into other, less toxic products. To this end, gulf toadfish were exposed to 3.8 mM 15NH(4)Cl in seawater for 24 and 48 h. Liver, kidney, gill, brain and muscle samples were analyzed for distribution of 15N within the tissue and among various nitrogen-containing metabolites (ammonia, amino-N, glutamine-N, urea and protein). The data reported here show that the toadfish can indeed take up and metabolize ammonia. Analysis of individual metabolic products of ammonia indicates that the toadfish can convert this toxic chemical into other less toxic metabolites. Ammonia enrichment is significantly different over controls in the kidney, brain and muscle. Urea enrichment is most significant in the brain, with less significant enrichment occurring in the liver and muscle. While accumulation of ammonia into an amino acid pool was not a significant metabolic fate, protein synthesis was significantly enriched in all tissues (with the highest levels occurring in the gill) indicating that amino acid synthesis may be a pathway of ammonia detoxification en route to protein synthesis, and that environmental ammonia can be 'fixed' into protein. Finally, it was found that glutamine-N synthesis occurs at significant levels in the liver, brain and muscle.
 
Article
Juvenile rainbow trout, Oncorhynchus mykiss, were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE(2)) through injection (1, 10, 25 and 50 microg EE(2)/g fish/week) and via water exposure (1, 10 and 100 ng EE(2)/l). After seven (injection and water exposure) and 14 days (only for water exposure), blood and plasma vitellogenin concentrations were quantified using indirect endpoints, i.e. plasma alkaline-labile phosphorus (ALP), plasma protein and plasma calcium. In addition, the relative gonad (GSI) and liver weight (HSI) were recorded. Actual plasma vitellogenin concentrations were measured with an enzyme immunoassay. Only fish injected with 50 microg EE(2)/g fish had a significantly higher gonad weight. No concentration-dependent changes in the HSI were detected in fish exposed via the water, but a significant dose-dependent increase of the HSI was observed in fish injected with EE(2). Exposure of rainbow trout to EE(2) had a significant effect on all tested plasma parameters. Plasma protein, phosphoprotein and calcium concentrations were significantly higher after two weeks exposure to 100 ng EE(2)/l. Fish injected with 10, 25 and 50 microg EE(2)/g fish exhibited increased plasma protein concentrations after 1 week. Compared to the controls, plasma ALP and calcium levels were significantly higher in all injected fish. A significant and positive correlation was observed between all three plasma parameters and between these indirect parameters and the actual plasma vitellogenin concentrations. These findings indicate that both the plasma ALP and the plasma calcium assay have a similar sensitivity as that of available antibody-based assays (EIA), at least in EE(2) exposure studies, and thus these assays can provide a rapid, simple and cost-effective alternative to available immunoassays.
 
Article
The effects of in vivo exposure to a natural and synthetic estrogen upon three hepatic phase II enzyme pathways involved in cellular protection against reactive intermediates were investigated in the largemouth bass (Micropterus salmoides). The pathways analyzed included glutathione S-transferases (GST), glutathione (GSH) biosynthesis and NAD(P)H-dependent quinone reductase (QR). Following exposure to 17-beta estradiol (E2, a model natural estrogen; 2 mg/kg, i.p.) or 4-nonylphenol (NP, a model synthetic estrogen; 5 mg/kg and 50 mg/kg, i.p.), serum vitellogenin concentrations in male fish were markedly increased. Exposure to E2 did not affect steady-state GST-A mRNA expression, although GST catalytic activity toward 1-chloro 2,4-dinitrobenzene (CDNB) was elevated at 48 h post-injection. In addition, the rates of bass liver GST-4-hydroxy-2-nonenal (GST-4HNE) conjugation were elevated by E2 exposure at all timepoints. In contrast, exposure to NP decreased steady-state GST-A mRNA levels, but did not alter GST catalytic activities. Hepatic GSH levels were not significantly affected by exposure to either compound, although a trend towards increased GSH biosynthesis was observed with both compounds. Although bass liver quinone reductase catalyzed 2,6-dichloroindophenol (DCP) reduction, unlike in rodents, these catalytic activities were not inhibited by dicoumarol. Exposure to 5 mg/kg NP significantly increased hepatic QR activities. Collectively, our data suggest that exposure to E2 or NP alters the ability of largemouth bass to biotransform environmental chemicals through glutathione S-transferase and quinone reductase catalytic pathways.
 
Article
Juvenile male western mosquitofish (Gambusia affinis) were exposed to different concentrations of 17alpha-ethynyl estradiol (EE2) in the diet during the period of sexual maturation. A clear inhibiting influence of EE2 on sexual development was apparent. The proportion of males in each treatment group that failed to complete gonopodial development during the 150-day observation period increased significantly with EE2 concentration. There were significant nonlinear trends toward shorter gonopodia in groups exposed to higher EE2 concentrations. Vitellogenin (VTG) was detectable in the blood of all fish exposed to 1.0 or more micro/g food and the concentration increased dramatically with increasing EE2 exposure. A significant negative association was seen between EE2 concentration and spermatophore counts. This study has demonstrated deleterious effects of EE2 exposure on sexual maturation and several indirect measures of reproductive fitness. It supports the biological relevance of vitellogenin in the blood and reduced gonopodium length as biomarkers for estrogen exposure and endocrine disruption in mosquitofish.
 
Article
Male Chinese loaches (Misgurnus anguillicaudatus) were exposed to 17beta-estradiol (E2) in the ambient water at nominal concentrations of 0.5, 1, 5 and 10 mug/L continuously for 8 weeks using a semi-static system. The gonadosomatic index (GSI), total plasma protein, total plasma calcium, magnesium and zinc, and plasma vitellogenin (Vtg) were determined as test endpoints. Our results indicate no significant changes in the GSI between exposed and control groups (P<0.05). All exposure concentrations of E2 dramatically induced the production of Vtg within 7 days. The vitellogenic response of male loach was time- and dose-dependent. At higher concentrations of E2 (5 and 10 microg/L), total plasma protein, total plasma calcium, and total plasma magnesium presented a significant time- and dose-dependent increase, and the three parameters were significantly correlated with plasma Vtg. We conclude that Chinese loaches are sensitive to estrogenic compounds and may be chosen as potential sentinel species in field and laboratory studies. In addition, total plasma protein, total plasma calcium, and total plasma magnesium can be used as indirect indicators to predict Vtg levels in estrogenized Chinese loach.
 
Article
The potential for contaminants to alter lipid or cholesterol dynamics in fish is rarely investigated and may include critical physiological endpoints that are impacted by exposure to endocrine-active substances. The current study investigated plasma and tissue lipid dynamics over a period of recrudescence in goldfish, while also examining the potential for beta-sitosterol (beta-sit), a phytosterol and 17beta-estradiol (E(2)), an endogenous estrogen, to alter lipid homeostasis. Goldfish were exposed to 0 microg/g (no chemical; control), 200 microg/g beta-sit (72.3% sitosterol mixture) or 10 microg/g 17beta-estradiol (E2) via Silastic implants for a period of five months. Plasma lipids peaked in control fish coincident with maximum liver size, while gonadal cholesterol concentration was highest concomitant with maximum gonad size. Plasma lipid concentrations were highly affected by E2 but not beta-sit exposure; E2 elevated total cholesterol (p<0.001) and triglyceride (TG; p<0.001) and decreased high-density lipoprotein (HDL) concentration (p<0.001) in male fish. Tissue cholesterol concentrations were minimally affected by beta-sit exposure, while hepatic cholesterol concentrations were increased in E2 exposed females (p=0.041), indicating elevated liver lipogenesis in response to E2, but not beta-sit, exposure. The present study demonstrates differential effects by beta-sit and E2 on plasma lipoprotein profile and TG concentration and indicates estrogen-specific effects on hepatic lipid metabolism during gonadal development.
 
Article
DNA hydrolysis caused by venoms of 17 species of snakes was studied by different methodologies. Endonucleolytic activity was tested by incubation of the venoms with the plasmid pBluescript and subsequent visualization of the electrophoretic patterns in 1% agarose gels stained with ethidium bromide. DNA was sequentially degraded, from supercoiled to opened circle, to linear form, in a concentration dependent manner. The highest hydrolytic activity was observed in Bothrops (B.) neuwiedii and Naja (N.) siamensis venoms. Exonucleolytic activity was analyzed on pBluescript digested with SmaI or EcoRI. All venoms caused complete hydrolysis after 2 h of incubation. SDS-PAGE analysis in gels containing calf thymus DNA showed that the hydrolytic bands were located at approximately 30 kDa. DNA degradation was studied by radial hydrolysis in 1% agarose gels containing calf thymus DNA plus ethidium bromide and visualized by UV light. Venom of B. neuwiedii showed the highest activity whereas those of B. ammodytoides and Ovophis okinavensis (P<0.05) showed the lowest activity. Antibodies against venom of B. neuwiedii or N. siamensis neutralized the DNAse activity of both venoms. In conclusion, venom from different snakes showed endo- and exonucleolytic activity on DNA. The inhibition of DNA hydrolysis by EDTA and heterologous antibodies suggests similarities in the structure of the venom components involved.
 
Article
Effects of estradiol-17beta (E2) and 5alpha-dihydrotestosterone (DHT) on the production of vitellogenin (Vg), insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) were examined in vitro using primary hepatocyte culture of the tilapia. Estradiol produced a significant and concentration-related stimulation of Vg release and concomitant, concentration-related reduction in IGF-I mRNA expression in both male and female hepatocytes. In male hepatocytes, DHT significantly increased IGF-I expression, whereas DHT inhibited IGF-I expression and stimulated Vg release in female hepatocytes. Estradiol treatment significantly reduced the release of 25 kDa IGFBP, while stimulating the release of 30 kDa IGFBP from male hepatocytes. In female hepatocytes, E2 significantly increased both 25 and 30 kDa IGFBPs. In male hepatocytes, DHT significantly reduced 25 kDa IGFBP without affecting 30 kDa IGFBP. Conversely, DHT treatment of hepatocytes from female fish significantly increased both the 25 and 30 kDa IGFBPs. The different growth rates observed between male and female tilapia may be a result of gonadal steroid hormones eliciting direct and antagonistic effects on production of IGF-I (growth) and Vg (reproduction) in the liver. Indeed, the different growth patterns likely result from a difference in the sensitivity of male and female hepatocytes to gonadal steroid hormones. These results also indicate direct effects of gonadal steroid hormones on production of IGFBPs, which may play a role in regulating IGF-I mediated growth.
 
Article
This study investigated the effects of different doses of 17-beta-estradiol (E(2)) in Rhamdia quelen. Groups of males exposed to different doses of E(2) (0.1 mg kg(-)(1), 1 mg kg(-)(1) and 10 mg kg(-)(1)) were compared with non-exposed male and female fish groups. Among the considered biomarkers, no significant differences were observed for micronuclei test, reduced glutathione concentration and lipid peroxidation. All E(2)-treated individuals had decreased glutathione S-transferase activity. Increased catalase and superoxide dismutase activities, increased vitellogenin expression and decreased metallothionein concentration were observed in males treated with the highest dose. Liver of all test groups showed necrotic areas, but cytoplasm vacuolization was again found only in the individuals exposed to highest dose. E(2) causes deleterious hepatic effects to R. quelen, and vitellogenin expression, catalase and superoxide dismutase activity and metallothionein concentration represent appropriate biomarkers for studying E(2) effects. Additionally, the response of some biomarkers was similar in males exposed to E(2) and unexposed females, and therefore exposure to endocrine disruptors may cause consequences for fish populations.
 
Article
The time- and dose-dependent transcriptional and translational expression of biomarker genes in nonylphenol (NP) and estradiol-17beta (E(2)) treated juvenile rainbow trout is reported. Fish were exposed to NP (1, 5 and 25 mg/kg) and E(2) (5 mg/kg) and killed at 2, 6, 12, 24, 48 and 72 h after exposure. The estrogen receptor (ER), vitellogenin (Vtg) and eggshell zona radiata protein (Zr-protein) gene expressions were analyzed in total liver RNA using Northern and slot hybridization with specific cDNA probes. Plasma Vtg and Zr-protein levels were evaluated using indirect ELISA. While Zr-protein gene showed an induction only at 24 h post-exposure, the plasma protein levels showed a time-dependent increase in the 25-mg NP treated group. Vtg transcripts showed an apparent time-dependent increase without a concomitant increase in protein levels in the 25-mg NP treated fish. Time-dependent increases in Vtg and Zr-protein gene expressions without the corresponding increases in ER gene transcription was observed in E(2)-treated fish at 2, 6 and 12 h post-exposure. Induction of ER gene transcripts was observed from 24 h and did not change significantly at 48 and 72 h. In the E(2)-treated fish, induction of plasma Vtg levels was observed at 48 and 72 h, while plasma Zr-protein was induced at 24, 48 and 72 h, after exposure. We conclude that the E(2)- and NP-induced Vtg and Zr-protein gene expressions at the early time intervals after exposure are not dependent on increase in the transcriptional activity of the ER gene and that Vtg and Zr-protein gene transcriptions require only basal or minimal ER concentration, in addition to other mechanisms.
 
Article
The mechanisms of action of an estrogenic chemical have been examined in a viviparous fish the eelpout (Zoarces viviparus), by identification of an upregulated estrogenic pathway--the induction of hepatic estrogen receptor mRNA, hepatic estrogen binding activity and plasma vitellogenin. A relative quantitative RT-PCR assay has been established to measure hepatic estrogen receptor alpha (ER) mRNA levels in eelpout. Assay conditions were optimised using control and induced samples to ascertain its applicability in the actual working range of ER mRNA concentrations. beta-Actin was co-amplified and used as an internal standard. Time-course effects of water exposure to 0.5 microg/L 17beta-estradiol (E(2)) and 25 microg/L of the xeno-estrogen 4-tert-octylphenol (4-tert-OP) on ER mRNA levels in the male eelpout was examined. After 48 h of exposure, ER transcripts were induced 15-fold and 6-fold in the E(2)- and OP-treated fish, respectively. This difference, however, was not apparent after 1 week of exposure, when similar high levels of ER mRNA were present in both groups (20-fold induction). This indicates that the estrogenic capacity of 4-tert-OP increases with exposure time. The effect of treatment was also evaluated by examining the induction of specific E(2) binding capacity in hepatic cytosolic extracts and by measuring vitellogenin in plasma. Both parameters were also induced by the treatments, but later in the time course. The measurement of ER mRNA by the RT-PCR assay showed to be the most sensitive method for the detection of estrogenic responses in eelpout.
 
Article
To determine the critical stage of zebrafish development where exposure to xenoestrogens can affect sex ratio and vitellogenin induction, zebrafish (Danio rerio) were exposed to 17alpha-ethinylestradiol (actual concentration 15.4+/-1.4 ng EE2/l) during early development: from fertilisation to hatch; hatch to 10 days post hatch (dph); 10-20 dph; 20-30 dph; 20-40 dph; 20-60 dph; fertilisation to 25 dph; or hatch to 60 dph. Vitellogenin was measured in whole body homogenate 30 dph by ELISA and sex ratio was determined 60 dph by histological examination of the gonads. All exposure periods significantly induced vitellogenin synthesis and affected the sex differentiation leading to development of ovo-testis or complete feminisation of the exposed fish depending on exposure period. Complete sex reversal was obtained in groups exposed from 20 to 60 dph and hatch to 60 dph. The half-life for degradation of vitellogenin was calculated. Juvenile zebrafish were exposed to 15.4+/-1.4 ng EE2/l (actual concentration) from fertilisation to 25 dph and transferred to clean water, after which weekly measurements of vitellogenin concentration in whole body homogenate were performed until day 46 post hatch. The half-life of vitellogenin was 2.4 days.
 
Article
The equilibrium binding parameters of the benzodiazepine antagonist [3H]Ro 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a]-1,4 benzodiazepine) were evaluated in brain membranes of the saltwater teleost fish, Mugil cephalus. To test receptor subtype specificity, displacement studies were carried out by competitive binding of [3H]Ro 15-1788 against six benzodiazepine receptor ligands, flunitrazepam [5-(2-fluoro-phenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one], alpidem [N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-acetamide], zolpidem [N,N-6 trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate], and beta-CCM (methyl beta-carboline-3-carboxylate). Saturation studies showed that [3H]Ro 15-1788 bound saturatably, reversibly and with a high affinity to a single class of binding sites (Kd value of 1.18-1.5 nM and Bmax values of 124-1671 fmol/mg of protein, depending on brain regions). The highest concentration of benzodiazepine recognition sites labeled with [3H]Ro 15-1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. The rank order of displacement efficacy of unlabelled ligands observed suggested that central-type benzodiazepine receptors are present in one class of binding sites (Type I-like) in brain membranes of Mugil cephalus. Moreover, the uptake of 36Cl- into M. cephalus brain membrane vesicles was only marginally stimulated by concentrations of GABA that significantly enhanced the 36Cl- uptake into mammalian brain membrane vesicles. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.
 
Article
Stress protein (heat shock protein, hsp70) response is involved in protecting organisms from the detrimental effects of environmental stressors, such as radiation and high temperatures. Tropical chitons can briefly tolerate high temperatures. However, they minimize the effects of elevated temperature during daylight hours and periods of tidal air exposure by remaining in rocky intertidal microhabitats along the shoreline of tropical waters. To study the natural variability of the hsp70 level, individuals of the polyplacophoran species Acanthopleura granulata Gmelin, 1791 were sampled every 4 h on two days in spring of 1999. Hsp70 levels were separately measured in the supernatant of the intestinal tract and foot muscle homogenates with a standardized immunoassay. The hsp70 level in the intestinal tract was highest in the early morning, decreased during the mid-morning hours and dropped to a comparatively low level in the afternoon, before increasing again during the night. The stress protein level in the foot muscle followed the daily air temperature curve with a time delay of a few hours, reaching the highest level in the afternoon and the lowest level in the early morning. The stress protein response can be interpreted as a sign of heat tolerance development and may play a role in allowing A. granulata to tolerate the temperature variability typical of its intertidal habitat.
 
Article
Adult male zebrafish (Danio rerio) were exposed to 17beta-estradiol (E2) or 17alpha-ethinylestradiol (EE2) in flow-through systems for 8 days. This was done to compare the sensitivity of the estrogen inducible vitellogenin (Vtg) biomarker system of this proposed OECD test guideline species to other relevant test species. Vtg was quantified in whole body homogenate by a species-specific ELISA. Actual water concentrations of E2 and EE2 were quantified by LC-MS, with detection limits of 1.0 and 0.6 ng/l, respectively. Vtg induction (LOEC) occurred in whole body homogenate at actual water concentrations of 21 ng E2/l and 3.0 ng EE2/l, respectively. As an alternative to the ANOVA approach, the relationship between the percentage of responding fish (Vtg) and the external E2 or EE2 concentration was determined by logistic regression analysis. Based on the regression analysis, EC-values could be determined: EC10, EC50 and EC90 were 15.4, 41.2 and 67.1 ng E2/l, respectively and 0.92, 2.51 and 4.09 ng EE2/l, respectively. Comparisons of these response limits to corresponding values for rainbow trout (Oncorhynchus mykiss), fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes) revealed the zebrafish as a sensitive test species.
 
Article
Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.
 
Article
Androgens originating from pulp mill processing, sewage treatment facilities and agricultural activities have the potential for discharge into aquatic receiving environments. To assess androgen effects on reproductive endocrine status in fish in estuarine environments, male and female adult northern mummichog (Fundulus heteroclitus macrolepidotus) were exposed to an aromatizable androgen (17α-methyltestosterone; MT) and a non-aromatizable androgen (5α-dihydrotestosterone; DHT) in a short-term reproductive endocrine bioassay. Fish were nominally exposed to 10μg/L or 100μg/L DHT, or 0.1μg/L or 1μg/L MT for 14days during gonadal recrudescence. Actual concentrations of androgens, as measured by enzyme immunoassay (EIA), were 10-49% of nominal MT 0.1, 3-6% of nominal MT 1, 5-10% of nominal DHT 10 and 3-25% of nominal DHT 100. Female mummichog were impacted to a greater degree by androgen exposure, with increased plasma testosterone (T) at 100μg/L DHT, depressed plasma 17β-estradiol (E2) at both DHT concentrations and at 1μg/L MT, as well as depressed in vitro E2 at both MT concentrations and 100μg/L DHT. Males had depressed plasma T in the 10μg/L DHT treatment and depressed in vitro 11-ketotestosterone production for both MT concentrations and 10μg/L DHT. Ovarian aromatase gene expression was induced in females exposed to 1μg/L MT. DHT at 100μg/L increased hepatic vitellogenin-1 (VTG1) expression in males and depressed VTG1 expression in females. The range of responses between sexes and among species provides evidence for modes of actions and potential impacts of androgens in aquatic receiving environments. Copyright © 2015. Published by Elsevier Inc.
 
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Chris Wood
  • University of British Columbia
François Gagné
  • Environment and Climate Change Canada
Jae-Seong Lee
  • Sungkyunkwan University
Colin Janssen
  • Ghent University
Adalto Bianchini
  • Universidade Federal do Rio Grande (FURG)