Showing papers in "Comparative Biochemistry and Physiology C-toxicology & Pharmacology in 2008"

Some insights into energy metabolism for osmoregulation in fish.

Abstract: A sufficient and timely energy supply is a prerequisite for the operation of iono- and osmoregulatory mechanisms in fish. Measurements of whole-fish or isolated-gill (or other organs) oxygen consumption have demonstrated regulation of the energy supply during acclimation to different osmotic environments, and such regulation is dependent on species, the situation of acclimation or acclimatization, and life habits. Carbohydrate metabolism appears to play a major role in the energy supply for iono- and osmoregulation, and the liver is the major source supplying carbohydrate metabolites to osmoregulatory organs. Compared with carbohydrates, the roles of lipids and proteins remain largely unclear. Energy metabolite translocation was recently found to occur between fish gill ionocytes and neighboring glycogen-rich (GR) cells, indicating the physiological significance of a local energy supply for gill ion regulatory mechanisms. Spatial and temporal relationships between the liver and other osmoregulatory and non-osmoregulatory organs in partitioning the energy supply for ion regulatory mechanisms during salinity challenges were also proposed. A novel glucose transporter was found to specifically be expressed and function in gill ionocytes, providing the first cue for investigating energy translocation among gill cells. Advanced molecular physiological approaches can be used to examine energy metabolism relevant to a particular cell type (e.g., gill ionocytes), and functional genomics may also provide another powerful approach to explore new metabolic pathways related to fish ion regulation.

Toxicity and effects of a glyphosate-based herbicide on the Neotropical fish Prochilodus lineatus.

Abstract: The toxicity of Roundup, a glyphosate-based herbicide widely used in agriculture, was determined for the Neotropical fish Prochilodus lineatus. The 96 h-LC(50) of Roundup was 13.69 mg L(-1), indicating that this fish is more sensitive to Roundup than rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). These differences should be considered when establishing criteria for water quality and animal well-being in the Neotropical region. Short-term (6, 24 and 96 h) toxicity tests were then performed to evaluate the effects of sub-lethal concentrations of the herbicide (7.5 and 10 mg L(-1)) to P. lineatus. Roundup did not interfere with the maintenance of the ionic balance and there was no significant alteration in plasma cortisol levels in Roundup-exposed fish. However an increase in plasma glucose was noted in fish exposed to 10 mg L(-1) of the herbicide, indicating a typical stress response. Catalase liver activity also showed an increase in fish exposed to 10 mg L(-1) of the herbicide, suggesting the activation of antioxidant defenses after Roundup exposure. In addition, Roundup induced several liver histological alterations that might impair normal organ functioning. Therefore, short-term exposure to Roundup at subletal concentrations induced biochemical, physiological and histological alterations in P. lineatus.

Comparative approaches to understand metal bioaccumulation in aquatic animals

Abstract: Over the past decades, comparative physiology and biochemistry approaches have played a significant role in understanding the complexity of metal bioaccumulation in aquatic animals. Such a comparative approach is now further aided by the biokinetic modeling approach which can be used to predict the rates and routes of metal bioaccumulation and assist in the interpretation of accumulated body metal concentrations in aquatic animals. In this review, we illustrate a few examples of using the combined comparative and biokinetic modeling approaches to further our understanding of metal accumulation in aquatic animals. We highlight recent studies on the different accumulation patterns of metals in different species of invertebrates and fish, and between various aquatic systems (freshwater and marine). Comparative metal biokinetics can explain the differences in metal bioaccumulation among bivalves, although it is still difficult to explain the evolutionary basis for the different accumulated metal body concentrations (e.g., why some species have high metal concentrations). Both physiological/biochemical responses and metal geochemistry are responsible for the differences in metal concentrations observed in different populations of aquatic species, or between freshwater and marine species. A comparative approach is especially important for metal biology research, due to the very complicated and potentially variable physiological handling of metals during their accumulation, sequestration, distribution and elimination in different aquatic species or between different aquatic systems.

Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae).

Abstract: Bacillus thuringiensis is one of the most widely used sources of biorational pesticides, as well as a key source of genes for transgenic expression to provide pest resistance in plants. In this study the effect of Bacillus thuringiensis ssp. galleriae (Bt) infection on the activity of superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), concentrations of oxidated and reduced thiols (RSSR/RSH) and malondialdehyde (MDA) was tested in the midgut of Galleria mellonella larvae. We found that Bt infection resulted in increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio the first day after inoculation. However, catalase activity decreased on the first and following days after bacterial infection by Bt. Our results confirm the hypothesis that Bt infection increases the level of oxidative stress in the larval midgut. In light of this study, it seems possible that oxidative damage contributes to cell death in the midgut during bacteriosis.

The neurobiology of social attachment: A comparative approach to behavioral, neuroanatomical, and neurochemical studies

Abstract: The formation and maintenance of social bonds in adulthood is an essential component of human health. However studies investigating the underlying neurobiology of such behaviors have been scarce. Microtine rodents offer a unique comparative animal model to explore the neural processes responsible for pair bonding and its associated behaviors. Studies using monogamous prairie voles and other related species have recently offered insight into the neuroanatomical, neurobiological, and neurochemical underpinnings of social attachment. In this review, we will discuss the utility of the microtine rodents in comparative studies by exploring their natural history and social behavior in the laboratory. We will then summarize the data implicating vasopressin, oxytocin, and dopamine in the regulation of pair bonding. Finally, we will discuss the ways in which these neurochemical systems may interact to mediate this complex behavior.

Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure.

Abstract: Cadmium (Cd) is one of the most toxic heavy metal pollutants in the aquatic environment and can induce the formation of reactive oxygen species (ROS) that cause oxidative stress. In present study, we cloned catalase (CAT) and glutathione peroxidase (GPX) cDNA, and investigated its time- and dose-related effects of three Cd concentrations (0.01, 0.05 or 0.1 ppm) on mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), CAT, GPX) in the gill and changes enzyme levels in the hemolymph of the Pacific oyster, Crassostrea gigas. The cDNA indentified encoded proteins of 516 and 244 amino acids corresponding to CAT and GPX, respectively. BLAST analysis from other species indicated that the residues essential to the enzymatic function of CAT and GPX proteins of C. gigas are highly conserved. Cd treatment significantly increased antioxidant enzyme mRNA expression in the gill in a time- and dose-dependent manner. The mRNA expression at 0.1 ppm Cd concentration increased up to 3 days (CAT, GPX) or 7 days (SOD) and then decreased by 7 days (CAT, GPX) or 11 days (SOD). Aspartate aminotransferase, alanine amintransferase and hydrogen peroxide (H2O2) concentrations levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results suggest that antioxidant enzymes play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.

Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas.

Abstract: Cadmium (Cd) is a widespread nonessential heavy metal that enters the aquatic environment as a result of natural processes and human activities such as wastewater production, agriculture, and mining. To determine the effects of Cd on organisms, we investigated its time- and dose-related effects on mRNA levels of heat shock protein 90 (HSP90) and metallothionein (MT) in the gill and digestive gland and changes enzyme levels in the hemolymph of the Pacific oyster Crassostrea gigas. Full-length HSP90 cDNA was isolated from C. gigas by rapid amplification of cDNA end (RACE) techniques and found to contain 2154 nucleotides, including an open reading frame, and was predicted to encode a protein of 717 amino acids. BLAST analysis indicated that the HSP90 gene of C. gigas shared high homology with known HSP90 genes of other mollusks. The expression of HSP90 mRNA increased significantly with exposure to 0.01 ppm Cd for 11 days or 0.05 or 0.1 ppm Cd for 7 days. The expression of MT mRNA increased significantly with exposure to 0.01, 0.05, or 0.1 ppm Cd for 11 days. Glutamate oxaloacetate and glutamate pyruvate levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results indicate that HSP90 and MT play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.

Antioxidant defenses and biochemical changes in pacu (Piaractus mesopotamicus) in response to single and combined copper and hypoxia exposure.

Abstract: The effect of combined-factors (hypoxia+copper) on the biochemical parameters and antioxidant defenses were studied in the neotropical fish Piaractus mesopotamicus. Fish were exposed for 48 h to 0.4 mg Cu(2+) L(-1) (0.4Cu), hypoxia=50 mm Hg (Hpx), and 0.4 mg Cu(2) L(-1)+hypoxia=50 mm Hg (0.4CuHpx). The exposure to 0.4Cu increased the reactive oxygen species (ROS) in the liver, accompanied by increases in superoxide dismutase (SOD) and decreases in catalase (CAT) activity, showing the influence of copper in this protection. The exposure to Hpx decreased the activity of glutathione peroxidase (GSH-Px) and CAT. Exposure to a combined-factor caused an increase in the ROS production followed by an increase in SOD and a decrease in GSH-Px and CAT. At 0.4Cu, fish presented a reduction in CAT, while in Hpx decreases in SOD, CAT and GSH-Px were observed in red muscles. Single-factors were insufficient to cause ROS production. In combined-factors, increased ROS formation and decreased SOD, CAT and GSH-Px were observed. RBC increased in all groups, but only under combined-factors was there an increase in hemoglobin. Copper plasma concentration increased in groups exposed to copper. Na(+)/K(+)-ATPase activity in gills decreased in 0.4Cu and 0.4CuHpx, and increased in Hpx. Metallothionein concentration in gills increased under combined-factors. Combined-factors caused significant disturbances in the antioxidant defenses and biochemical parameters than single-factors.

Theoretical considerations underlying Na(+) uptake mechanisms in freshwater fishes.

Abstract: Ion and acid-base regulating mechanisms have been studied at the fish gill for almost a century. Original models proposed for Na(+) and Cl(-) uptake, and their linkage with H(+) and HCO(3)(-) secretion have changed substantially with the development of more sophisticated physiological techniques. At the freshwater fish gill, two dominant mechanisms for Na(+) uptake from dilute environments have persisted in the literature. The use of an apical Na(+)/H(+) exchanger driven by a basolateral Na(+)/K(+)-ATPase versus an apical Na(+) channel electrogenically coupled to an apical H(+)-ATPase have been the source of debate for a number of years. Advances in molecular biology have greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This review will focus on the recent molecular advances for Na(+) uptake in freshwater fish. Emphasis will be placed on thermodynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermodynamic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner.

Neuropharmacology and behavior in planarians: Translations to mammals

Abstract: Planarians are the simplest animals to exhibit a body plan common to all vertebrates and many invertebrates, characterized by bilateral rather than radial symmetry, dorsal and ventral surfaces, and a rostrocaudal axis with a head and a tail, including specialized sense organs and an aggregate of nerve cells in the head. Neurons in planarian more closely resemble those of vertebrates than those of advanced invertebrates, exhibiting typical vertebrate features of multipolar shape, dendritic spines with synaptic boutons, a single axon, expression of vertebrate-like neural proteins, and relatively low spontaneously generated electrical activity. Here we report the most relevant contribution to the knowledge of the neuropharmacology of planarians, with particular reference to the behavioral consequences of the exposure to drugs acting on neural transmission. Neurochemical and histochemical data indicate the presence of several neurotransmitter-receptor systems in planarians. Moreover, a variety of experimental studies characterized specific behavioral patterns of these animals following the exposure to drugs acting on neural transmission. There is also evidence of the interactions between discrete neurotransmitter-receptor systems in modulating behavior in planarians. Finally, the model has proved efficacy for investigating the neurotoxicology of the dopamine neurons, and for the initial screening of the neuroprotective potential of drugs. In conclusion, these findings indicate that interactions between discrete neurotransmitter-receptor systems occur very early along phylogeny, although they may have evolved from very fundamental behaviors, such as motor activity in planarian, to more complex and integrated functions in vertebrates.

Endocrine-related reproductive effects in molluscs

Abstract: Research on endocrine disruption has been a major topic of the past decade. Although most studies concentrated on vertebrate species, invertebrates are now gaining more attention. In particular, data on molluscs is increasing. One of the best-documented and more relevant examples of endocrine disruption is the imposex phenomenon affecting some gastropod species. But the increasing interest is also due to the fact that molluscs, especially bivalves, are good bioindicators used for decades in environmental studies and that progress have been made in the understanding of the physiology and endocrinology of some mollusc species. Recent results suggest that molluscs can be adversely affected by compounds that alter their reproduction and that vertebrate-type sex-steroids metabolism or mechanism of action could be involved in these effects. Nevertheless, the endocrine system of molluscs appears to be dissimilar in many aspects to those of vertebrates and sex-steroids might not have the same importance in all mollusc species. This diversity constitutes an important opportunity to examine and understand new and alternative mechanisms for endocrine disruption.

Vanadium and cadmium in vivo effects in teleost cardiac muscle: metal accumulation and oxidative stress markers.

Abstract: Several biological studies associate vanadium and cadmium with the production of reactive oxygen species (ROS), leading to lipid peroxidation and antioxidant enzymes alterations. The present study aims to analyse and compare the oxidative stress responses induced by an acute intravenous exposure (1 and 7 days) to a sub-lethal concentration (5 mM) of two vanadium solutions, containing different vanadate n -oligomers ( n = 1–5 or n = 10), and a cadmium solution on the cardiac muscle of the marine teleost Halobatrachus didactylus (Lusitanian toadfish). It was observed that vanadium is mainly accumulated in mitochondria (1.33 ± 0.26 μM), primarily when this element was administrated as decameric vanadate, than when administrated as metavanadate (432 ± 294 nM), while the highest content of cadmium was found in cytosol (365 ±231 nM). Indeed, decavanadate solution promotes stronger increases in mitochondrial antioxidant enzymes activities (catalase: + 120%; superoxide dismutase: + 140%) than metavanadate solution. On contrary, cadmium increases cytosolic catalase (+ 111%) and glutathione peroxidases (+ 50%) activities. It is also observed that vanadate oligomers induce in vitro prooxidant effects in toadfish heart, with stronger effects induced by metavanadate solution. In summary, vanadate and cadmium are differently accumulated in blood and cardiac subcellular fractions and induced different responses in enzymatic antioxidant defence mechanisms. In the present study, it is described for the first time the effects of equal doses of two different metals intravenously injected in the same fish species and upon the same exposure period allowing to understand the mechanisms of vanadate and cadmium toxicity in fish cardiac muscle.

Effects of water chemistry variables on gill binding and acute toxicity of cadmium in rainbow trout (Oncorhynchus mykiss): A biotic ligand model (BLM) approach.

Abstract: This study investigated the short-term (3 h) cadmium binding characteristics of the gills, as well as the influence of various water chemistry variables [calcium, magnesium, sodium, pH, alkalinity and dissolved organic carbon (DOC)] on short-term gill accumulation and acute toxicity of cadmium in juvenile freshwater rainbow trout. The cadmium binding pattern revealed two types of cadmium binding sites in the gill: (i) saturable high affinity sites operating at a low range of waterborne cadmium concentration, and (ii) non-saturable low affinity sites operating at a higher range of cadmium concentration. Among the water chemistry variables tested, only calcium and DOC significantly reduced both gill accumulation and toxicity of cadmium. Interestingly, alkalinity (15-90 mg L(-1) as CaCO(3)) did not influence the gill cadmium accumulation but a significant increase in toxicity was recorded at a higher alkalinity level (90 mg L(-1)). Affinity constants (log K) for binding of competing cations (Cd(2+) and Ca(2+)) to the biotic ligand and for binding of Cd(2+) to DOC were derived separately from the 3 h gill binding tests and the 96 h toxicity tests. In general, the values agreed well, indicating that both tests targeted the same population of high affinity binding sites, which are likely Ca(2+) uptake sites on the gills. These parameters were then incorporated into a geochemical speciation model (MINEQL+) to develop a biotic ligand model for predicting acute toxicity of cadmium in trout. The model predictions exhibited a good fit with the measured toxicity data except for high alkalinity and pH.

Candidates for membrane progestin receptors—Past approaches and future challenges

Abstract: Progestins have a broad range of functions in reproductive biology. Many rapid nongenomic actions of progestins have been identified, including induction of oocyte maturation, modulation of reproductive signaling in the brain, rapid activation of breast cancer cell signaling, induction of the acrosomal reaction and hypermotility in mammalian sperm. Currently, there are three receptor candidates for mediating rapid progestin actions: (1) membrane progestin receptors (mPRs); (2) progestin receptor membrane components (PGRMCs); and (3) nuclear progestin receptors (nPRs). The recently-described mPR family of proteins has seven integral transmembrane domains and mediates signaling via G-protein coupled pathways. The PGRMCs have a single transmembrane with putative Src homology domains for potential activation of second messengers. The classical nPRs, in addition to having well defined transcriptional activity, can also mediate rapid activation of intracellular signaling pathways. However, details of the mechanisms by which these three classes of progestin receptors mediate rapid intracellular signaling and their subcellular localization remain unclear. In addition, mPRs, nPRs and PGRMCs exhibit overlapping expression and functions in multiple tissues, implying potential interactions during oocyte maturation, parturition, and breast cancer signaling in individual cells. However, the overwhelming majority of studies to date have focused on the functions of one of these groups of receptors in isolation. This review will summarize recent findings on the three major progestin receptor candidates, emphasizing the different approaches used, some experimental pitfalls, and current controversies. We will also review evidence for the involvement of mPRs and nPRs in one of the most well-characterized nongenomic steroid actions in basal vertebrates, oocyte maturation, and conclude by suggesting some future areas of research. Clarification of the controversies surrounding the identities and localization of membrane progestin receptors may help direct future research that could advance our understanding of rapid actions of steroids.

Cortisol response to acute stress in jundiá Rhamdia quelen acutely exposed to sub-lethal concentrations of agrichemicals

Abstract: Exposure to agrichemicals can have deleterious effects on fish, such as disruption of the hypothalamus–pituitary–inter-renal axis (HPI) that could impair the ability of fish to respond to stressors. In this study, fingerlings of the teleost jundia (Rhamdia quelen) were used to investigate the effects of the commonly used agrichemicals on the fish response to stress. Five common agrichemicals were tested: the fungicide — tebuconazole, the insecticide — methyl-parathion, and the herbicides — atrazine, atrazine + simazine, and glyphosate. Control fishes were not exposed to agrichemicals and standard stressors. In treatments 2–4, the fishes were exposed to sub-lethal concentrations (16.6%, 33.3%, and 50% of the LC50) of each agrichemical for 96 h, and at the end of this period, were subjected to an acute stress-handling stimulus by chasing them with a pen net. In treatments 5–7 (16.6%, 33.3%, and 50% of the LC50), the fishes were exposed to the same concentrations of the agrichemicals without stress stimulus. Treatment 8 consisted of jundias not exposed to agrichemicals, but was subjected to an acute stress-handling stimulus. Jundias exposed to methyl-parathion, atrazine + simazine, and glyphosate presented a decreased capacity in exhibiting an adequate response to cope with stress and in maintaining the homeostasis, with cortisol level lower than that in the control fish (P

Antioxidant defence enzyme activities in hepatopancreas, gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube ☆

Abstract: The aim of our study was to determine the activity of antioxidant defence (AD) enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the hepatopancreas, the gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube and to compare tissue specificities of investigated enzymes. Our results indicated that both specific and total SOD activities in the hepatopancreas were lower compared to the gills and muscle. Total SOD activity in the gills was lower with respect to that in muscle. CAT and GSH-Px (both specific and total) activities were higher in the hepatopancreas compared to those in the gills and muscle. In the gills the specific and total GR activities were higher than in the hepatopancreas and muscle. The specific and total GST activities were higher in the hepatopancreas compared with the gills and muscle. Our study represents the first comprehensive report of AD enzymes in tissues of O. limosus caught in the River Danube. The noted tissue distributions of the investigated AD enzyme activities most likely reflected different metabolic activities and different responses to environmental conditions in the examined tissues.

Toxicological effects of methylmercury on walleye (Sander vitreus) and perch (Perca flavescens) from lakes of the boreal forest.

Abstract: Biochemical and physiological responses of walleye (Sander vitreus) and perch (Perca flavescens) were studied in four Canadian boreal forest lakes representing a mercury (Hg) exposure gradient The aim of this study was to assess the effects of Hg and methylmercury (MeHg) on the general physiological condition of fish as well as to gauge the relationship between MeHg and the glutathione (GSH) system in metal-contaminated and reference sites using a series of biomarkers Walleye from Lake Malartic had the highest liver MeHg concentrations, exhibited lower hepatosomatic indices (HSI) and lower glutathione S-transferase (GST) activity HSI was negatively related to liver total Hg concentrations in walleye (R2 = 033, n = 108, P

Modulation of gene expression in hibernating arctic ground squirrels

Abstract: We performed a broadscale screening of differential gene expression using both high-throughput bead-array technology and real-time PCR assay in brown adipose tissue, liver, heart, hypothalamus, and skeletal muscle in hibernating arctic ground squirrels, comparing animals sampled after two durations of steady-state torpor, during two stages of spontaneous arousal episodes, and in animals after they ended hibernation. Significant seasonal and torpor-arousal cycle differences of gene expression were detected in genes involved in glycolysis, fatty acid metabolism, gluconeogenesis, amino acid metabolism, molecular transport, detoxification, cardiac contractility, circadian rhythm, cell growth and apoptosis, muscle dystrophy, and RNA and protein protection. We observed, for the first time, complex modulation of gene expression during multiple stages of torpor-arousal cycles. The mRNA levels of certain metabolic genes drop significantly during the transition from late torpor to early arousal, perhaps due to the rapid turnover of mRNA transcripts resulting from the translational demands during thermogenesis in early arousal, whereas the mRNA levels of genes related to circadian rhythm, cell growth, and apoptosis rise significantly in the early or late arousal phases during torpor-arousal cycle, suggesting the resumption of circadian rhythm and cell cycle during arousal.

Dynamic changes in parameters of redox balance after mild heat stress in aged laying hens (Gallus gallus domesticus).

Abstract: In order to evaluate the metabolic responses of laying hens induced by high temperature at later laying stage, nine 60-wk-old laying hens (Gallus gallus domesticus) were employed in the present study. The hens were exposed to 32 degrees C for 21 d and blood samples were obtained before and at 1, 7, 14 and 21 d of heat exposure. The reactive oxygen species (ROS) formed in blood during heat exposure were estimated by the ex vivo spin-trapping method. Body temperature and plasma concentrations of glucose, urate, creatine kinase (CK), triiodothyronine (T(3)), thyroxine (T(4)), corticosterone (CORT), thiobarbituric acid reacting substances (TBARS), ferric/reducing antioxidant power (FRAP) and superoxide dismutase (SOD) activity were measured. Plasma levels of glucose, CK and CORT were not significantly influenced by heat exposure at any time point. The circulating concentrations of T(3) were decreased while plasma T(4) levels changed in the opposite way. The formation of ROS was significantly augmented by heat exposure in laying hens though the body temperature was not significantly altered. The enhanced enzymatic and non-enzymatic antioxidant systems acted in concert to alleviate the heat stress evoked oxidative damage.

Endocrine disrupting chemicals (EDC) with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: II. Effects on gonad histomorphology.

Abstract: A number of man-made chemicals has been shown to mimic endogenous hormones and to induce alterations of reproductive physiology in wild populations. Of particular importance are compounds that mimic estrogens and androgens (and their antagonists), because of their central role in reproductive function. In this study, male and female adult South African clawed toads ( Xenopus laevis) were exposed to ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT) and flutamide (FLU) as (anti)estrogenic and (anti)androgenic model compounds, respectively, at a concentration of 10 − 8 M, and to water from the river Lambro (LAM), a contaminated watercourse from Northern Italy. Potential disrupting effects on reproduction were studied by histological analyses of gonads. The strongest adverse effects were observed in EE2 and LAM exposed males, e.g. tubule mean diameter reduction, spermatogenic nest breakdown and interlobular wall thickening. In both groups, the occurrence of small oocytes within the seminiferous tubules was observed. In TAM and MDHT exposed females slight oocyte atresia and occurrence of spermatogenic nests were observed. In contrast to previous studies addressing the alteration of molecular biomarkers in the same experimental setup, histological analyses of gonads were very sensitive and indicated an adverse effect of water from Lambro River on reproductive physiology of X. laevis .

Transcriptional effects of PFOS in isolated hepatocytes from Atlantic salmon Salmo salar L.

Abstract: The main aim of the current in vitro experiment was to search for makers for PFOS exposure in isolated hepatocytes from Atlantic salmon Salmo salar, based on genes responding to PFOS exposure in other animals. Primary cell cultures of hepatocytes were exposed to four concentrations of PFOS (2.1-6.2-15.1-25.0 mg/L) for 24 and 48 h and the transcriptional levels of 12 genes encoding proteins known to respond to PFOS were quantified with real-time RT-PCR. The 12 examined genes were caspase 3B (apoptosis), GSH-Px and HSP70 (cellular stress), CYP1A, CYP3A, GST and UGT (P450 and phase II enzymes), acyl-CoA oxidase, PPARalpha, PPARbeta and PPARgamma (lipid metabolism) and Na(+)-K(+)-ATPase (ion regulation). Most of the studied genes responded in a dose-dependent manner to PFOS exposure, although the transcriptional differences in general where small with regard to fold change. Our results clearly suggest that PFOS exposure enhanced cellular stress in the examined cells, even though the exact mechanisms behind this stress remain unknown. The results from this in vitro experiment showed that genes known to be affected by PFOS exposure in other species also were induced in hepatocytes of Atlantic salmon, giving us the rationale to expand to testing the actual in vivo magnitude of effect in Atlantic salmon exposed to PFOS at doses usually seen in nature/diets.

Mercury toxicity in livers of northern pike (Esox lucius) from Isle Royale, USA

Abstract: Many laboratory studies have documented that mercury can be toxic to fish, but it is largely unknown if mercury is toxic to fish in their natural environments. The objective of our study was to investigate the toxic effects of mercury on northern pike (Esox lucius) at Isle Royale, Michigan. In 124 northern pike from eight inland lakes, concentrations of total mercury in skin-on fillets ranged from 0.069 to 0.622 μg/g wet mass (wet wt). Concentrations of total mercury in livers increased exponentially compared with concentrations in fillets, to a maximum of 3.1 μg/g wet wt. Methylmercury constituted a majority of the mercury in livers with total mercury concentrations 0.5 μg/g wet wt. Liver color (absorbance at 400 nm) varied among northern pike and was positively related to liver total mercury concentration. The pigment causing variation in liver color was identified as lipofuscin, which results from lipid peroxidation of membranous organelles. An analysis of covariance revealed lipofuscin accumulation was primarily associated with mercury exposure, and this association obscured any normal accumulation from aging. We also documented decreased lipid reserves in livers and poor condition factors of northern pike with high liver total mercury concentrations. Our results suggest (i) northern pike at Isle Royale are experiencing toxicity at concentrations of total mercury common for northern pike and other piscivorous fish elsewhere in North America and (ii) liver color may be useful for indicating mercury exposure and effects in northern pike at Isle Royale and possibly other aquatic ecosystems and other fish species.

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper.

Abstract: The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6+/-0.2 mg kg(-1) Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7+/-0.3 mg kg(-1) Cu), an experimental diet enhanced with Cu (135+/-4 mg kg(-1) Cu) or an experimental diet (7.7+/-0.3 mg kg(-1) Cu) whilst exposed to Cu in water (0.294+/-0.013 mg L(-1)). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.

Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensi.

Abstract: A hyaluronidase, named BmHYA1, was purified from the venom of Chinese red scorpion ( Buthus martensi ), using successive chromatography. The homogeneity of BmHYA1 was confirmed by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular mass of BmHYA1 was 48,696 Da determined by MALDI-TOF MS. The optimal temperature and pH of BmHYA1 were 50 °C and pH 4.5, respectively. It could be inhibited by DTT, Cu 2+ , Fe 3+ or heparin, but not Mg 2+ , Ca 2+ , reduced glutathione, l -cysteine or EDTA. The sequence of thirty N-terminal amino acids of BmHYA1 was obtained by Edman degradation, as TSADF KVVWE VPSIM CSKKF KICVT DLLTS; but no similarity was found to other venom hyaluronidases. Further, BmHYA1 can hydrolyze hyaluronan into relatively smaller oligosaccharides and modulate the expression of CD44 variant in the breast cancer cell line MDA-MB-231.

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus).

Abstract: Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.

Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium-dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha

Abstract: Glutathione S-transferases (GST) and glutathione peroxidases (GPx) are essential components of cellular detoxification systems. We identified GST and GPx transcripts in the freshwater bivalve Dreissena polymorpha, their full-length coding sequences were obtained by reverse-transcription PCR using degenerated primers followed by 5′ and 3′ RACE-PCR (rapid amplification of cDNA ends-PCR). The cDNA identified encoded proteins of 205 and 243 amino acids corresponding respectively to a pi-class GST and a selenium-dependent GPx. The comparison of the deduced amino acid sequences with GST and GPx from other species showed that the residues essential to the enzymatic function of these two proteins are highly conserved. We studied their expression pattern in the digestive gland, the gills and the excretory system of D. polymorpha. The results showed that pi-GST mRNA expression is higher in the digestive gland than in the gills or the excretory system. Se-GPx transcripts are expressed at high, medium and very low levels in the digestive gland, the excretory system and the gills, respectively.

Acute effects of diflubenzuron on the freshwater fish Prochilodus lineatus.

Abstract: The effects of diflubenzuron (DFB), an insecticide to control ectoparasites in fish farms, on muscle acetylcholinesterase (AChE), detoxifying and antioxidant enzymes, hematological and physiological parameters, and liver histopathology were evaluated in Prochilodus lineatus after 6, 24 and 96 h of exposure to 25 mg L − 1 of DFB. The insecticide caused a reduction in the number of erythrocytes and hemoglobin content after 96 h exposure, probably due to hemolysis. Hyperglycemic response indicated energy mobilization, and may have contributed to the increase in osmolarity after 96 h exposure to DFB. The induction of glutathione- S -transferase (GST) and catalase activities in liver pointed to the activation of xenobiotic metabolic pathways and antioxidant defenses. The decrease in muscle AChE at all experimental times showed that DFB is an AChE inhibitor. In addition, DFB induced hepatic alterations that might impair normal liver functions. These results show that DFB can cause health disorders in fish and further studies are required to better define its safe use in aquaculture.

Comparative fate of organohalogen contaminants in two top carnivores in Greenland: captive sledge dogs and wild polar bears.

Abstract: The limited knowledge and/or the inability to control physiological condition parameters that influence the fate of organohalogen contaminants (OHCs) has been the foremost confounding aspect in monitoring programs and health risk assessments of wild top predators in the Arctic such as the polar bear (Ursus maritimus). In the present comparative study, we used a potential surrogate Canoidea species for the East Greenland polar bear, the captive sledge dog (Canis familiaris), to investigate some factors that may influence the bioaccumulation and biotransformation of major chlorinated and brominated OHCs in adipose tissue and blood (plasma) of control (fed commercial pork fat) and exposed (fed West Greenland minke whale (Balaenoptera acutorostrata) blubber) adult female sledge dogs. Furthermore, we compared the patterns and concentrations of OHCs and their known or suggested hydroxylated (OH) metabolites (e.g., OH-PCBs) in sledge dogs with those in adipose tissue and blood (plasma) of East Greenland adult female polar bears, and blubber of their main prey species, the ringed seal (Pusa hispida). The two-year feeding regime conducted with sledge dogs led to marked differences in overall adipose tissue (and plasma) OHC residue accumulation between the control and exposed groups. Characteristic prey-to-predator OHC bioaccumulation dynamics for major PCB and PBDE congeners (patterns and concentrations) and biotransformation capacity with respect to PCB metabolite formation and OH-PCB retention distinguished, to some extent, captive sledge dogs and wild polar bears. Based on the present findings, we conclude that the use of surrogate species in toxicological investigations for species in the Canoidea family should be done with great caution, although they remain essential in the context of contaminants research with sensitive arctic top carnivore species such as the polar bear.

Shrimp thioredoxin is a potent antioxidant protein

Abstract: ARTICLE I NFO Thioredoxin (TRX) is a main component of the redox homeostasis machinery in the cell and it is required for ribonucleotide reductase function among others. In invertebrates, the redox balance is compromised during disease and changes in the physiological state and it is one of the components of the innate immune response. In this work, the shrimp (Litopenaeus vannamei) LvTRX cDNA was sequenced, cloned and over- expressed in bacteria to further characterize the function of the recombinant protein. LvTRX was able to reduce insulin disulfides and it was a better antioxidant compared to reduced glutathione and ascorbic acid, by means of the Trolox Equivalent Antioxidant Capacity (TEAC) assay. Interestingly, LvTRX contains aside of the canonical active site CXXC disulfide motif, one Cys (C73) residue in the interface of a putative dimer previously reported for human TRX. Using qRT-PCR, we found that shrimp LvTRX is mainly expressed in gills and pleopods; the variation of LvTRX mRNA upon hypoxia and re-oxygenation is not statistically significant. LvTRX stands as an important antioxidant that must be considered in future physiological and immune challenges studies.

Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity.

Abstract: Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout ( Oncorhynchus mykiss ) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2′,5′-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, K M and V max values for GSSG and NADPH were also determined for the enzyme. In addition, K i values and inhibition types were determined for GSH and NADP + . Additionally, inhibitory effects of metal ions (Cd + 2 , Cu + 2 , Pb + 2 , Hg + 2 , Fe + 3 and Al + 3 ) on glutathione reductase were investigated. K i constants and IC 50 values for metal ions were determined by Lineweaver–Burk graphs and plotting activity % vs. [I], respectively. IC 50 values of Cd + 2 ,Cu + 2 , Pb + 2 , Hg + 2 , Fe + 3 and Al + 3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the K i constants for Cd + 2 and Cu + 2 were 0.104 ± 0.001, 0.117 ± 0.001, respectively.