Effects of Cu on plasma cortisol and cortisol secretion by adrenocortical cells of rainbow trout (Oncorhynchus mykiss)

Fish are exposed to multiple stressors, often acting concurrently, in their environment. To evaluate the potential of Cu to act as a chemical stressor, rainbow trout (Oncorhynchus mykiss) were exposed to Cu (30 or 80 microg/l) for 30 days in the laboratory and they were subjected to a physical stressor (1 min air exposure) before sampling. Physiological stress indicators in the whole fish as well as cortisol secretion by adrenocortical cells in vitro were measured. Fish exposed to Cu had a lower condition factor, hepatosomatic index, plasma glucose, hepatic glycogen and gill Na(+)/K(+)-ATPase activity compared to controls. Exposure to Cu did not have an effect on basal plasma cortisol (fish sampled without air exposure stress) however, the air exposure-induced increase in plasma cortisol was lower in fish exposed to Cu. Cortisol secretion stimulated by ACTH in vitro was greater in adrenocortical cells isolated from fish exposed to Cu in vivo but in vitro exposure to Cu consistently impaired cortisol secretion. Our results indicate that Cu at high concentrations disrupts cortisol secretion through a direct toxic effect on adrenocortical cells while low concentrations resulting from a 30-day exposure to environmentally relevant Cu concentrations enhances cortisol secretion in response to ACTH in vitro.     

The effects of copper on the morphological and functional development of zebrafish embryos

Waterborne copper exposure can exert a variety of physiological effects in fish, including the disruption of sensory system function, which has wide-reaching implications for fish behaviour. In developing fish larvae, copper is known to affect key parameters, such as survival and growth and more recently has been shown to interfere with the octavolateral system. The present study aimed to take a combined view of morphological (e.g. length, yolk sac area) and functional (e.g. heart beat, behaviour) processes to understand the complex effect of copper on fish development. In the first of two experiments, zebrafish embryos were exposed to a range of copper concentrations (11-1000 microg l(-1)) from fertilisation for a 72 h period. The greatest mortality was seen between 5 and 24h post-fertilisation (hpf) and was more pronounced at the higher copper concentrations. Copper also had an inhibitory effect on hatching. Length and yolk sac area of individuals were recorded across treatments at 72 hpf and elevated copper was found to slow development. Individuals from the higher copper treatments had the fastest heart rates at 28 hpf suggesting that stress responses were induced in the embryos during copper exposure. In the second experiment, embryos were exposed in a similar manner to two copper concentrations, based on those from Experiment 1 that resulted in <50% mortality. At 120 hpf, embryos exposed to both copper concentrations possessed significantly fewer functional neuromasts, an effect which was associated with a reduced ability to orientate in a current. Therefore, although mortality at these copper concentrations was low initially, and then almost non-existent after 24 hpf, the inability of copper-exposed larvae to orientate in a water current as a result of lateral line dysfunction is likely to seriously compromise survival.     

Developmental lead exposure causes startle response deficits in zebrafish

Lead (Pb(2+)) exposure continues to be an important concern for fish populations. Research is required to assess the long-term behavioral effects of low-level concentrations of Pb(2+) and the physiological mechanisms that control those behaviors. Newly fertilized zebrafish embryos (<2h post fertilization; hpf) were exposed to one of three concentrations of lead (as PbCl(2)): 0, 10, or 30 nM until 24 hpf. (1) Response to a mechanosensory stimulus: Individual larvae (168 hpf) were tested for response to a directional, mechanical stimulus. The tap frequency was adjusted to either 1 or 4 taps/s. Startle response was recorded at 1000 fps. Larvae responded in a concentration-dependent pattern for latency to reaction, maximum turn velocity, time to reach V(max) and escape time. With increasing exposure concentrations, a larger number of larvae failed to respond to even the initial tap and, for those that did respond, ceased responding earlier than control larvae. These differences were more pronounced at a frequency of 4 taps/s. (2) Response to a visual stimulus: Fish, exposed as embryos (2-24 hpf) to Pb(2+) (0-10 μM) were tested as adults under low light conditions (≈ 60 μW/m(2)) for visual responses to a rotating black bar. Visual responses were significantly degraded at Pb(2+) concentrations of 30 nM. These data suggest that zebrafish are viable models for short- and long-term sensorimotor deficits induced by acute, low-level developmental Pb(2+) exposures.     

Toxic effects of celastrol on embryonic development of zebrafish (Danio rerio)

Celastrol is a terpenoid purified from Tripterygium wilfordii Hook F. As a natural product with pharmacological activities, this compound is a promising candidate for drug development. To provide more information about its toxicity for clinical trials, toxicity assessment of celastrol was conducted with zebrafish model in vivo. 1hour post-fertilization (hpf) embryos were treated with various concentrations of celastrol for 120h. Developmental phenotypes were observed and survival rates were recorded. The results showed that the hatching rates of embryos treated with 1.0μM or higher celastrol were significantly lower. Embryos exposed to 1.0μM celastrol had no blood flow in trunk vessels at 48hpf with a median effect concentration (EC(50)) of 0.94μM. At 72hpf serious edema in pericardial sac was observed in the surviving larvae (hatched from embryos treated with 1.5μM celastrol). Bent tails or hook-like tails were seen as 0.5μM celastrol and the EC(50) for tail malformation was 0.66 μM at 72hpf. The lethal effect of celastrol on zebrafish embryos was dose-dependent and the LC(50) values of celastrol on 1hpf embryos were approximately 1.40μM. These results indicate that celastrol affects the normal development of zebrafish embryo in μM concentrations.     

Effects of embryonic propofol exposure on axonal growth and locomotor activity in zebrafish

Prenatal propofol exposure induced neurotoxicity in the developing brains and led to persistent learning deficits in the offspring. Our goal was to use zebrafish to explore whether the decline in learning and memory was correlated with inhibition of neuronal growth after propofol exposure. Zebrafish embryos at 6 hours postfertilization (hpf) were exposed to control or 1, 2 or 4 μg/mL propofol until 48 hpf. Spontaneous locomotor activity and swimming behavior in response to dark-to-light photoperiod stimulation were studied in zebrafish larvae at 6 days postfertilization (dpf). The adaptability to repeated stimulation was used to indicate learning and memory function of larvae. Transgenic NBT line zebrafish was used to quantitate the effect of propofol on motor neuronal growth of embryos in vivo. Six dpf transgenic zebrafish larvae went through photoperiod stimulation after their neuronal length had been analyzed during the embryonic period. Our data indicate that embryonic exposure to 1, 2 and 4 μg/mL propofol had no adverse effect on spontaneous movement in zebrafish larvae, but 2 and 4 μg/mL propofol significantly impaired the learning and memory function of larvae. Moreover, propofol significantly inhibited axonal growth of motor neurons during the embryonic stage, which was correlated with learning and memory deficiency in larvae. Our findings demonstrate that the neuronal growth was correlated with learning and memory function, indicating the relevance of zebrafish as a new model to explore the mechanisms through which propofol induces long-term learning and memory impairment.     

The effect of 4-nonylphenol on semen quality, viability of gametes, fertilization success, and embryo and larvae survival in rainbow trout (Oncorhynchus mykiss)

The present study investigated in vivo and in vitro effects of environmental relevant concentrations of 4-nonylphenol (100-750 ng l(-1)) on the reproduction of rainbow trout (Oncorhynchus mykiss). To determine the effect of 4-nonylphenol on semen quality rainbow trout were exposed to three concentrations of 4-nonylphenol in a flow-through system during the spawning period (60 days). At an estimated 4-nonylphenol concentration of 750 ng l(-1) semen production was completely inhibited, at 280 and 130 ng l(-1) the semen production was significantly reduced in comparison to the control. Sperm density, sperm motility and sperm fertility were not affected. Also the development of embryos and larvae at the end of yolk sac stage was affected by 4-nonylphenol. At estimated 4-nonylphenol exposure levels of 280 and 750 ng l(-1) the percentage of eyed stage embryos was slightly but significantly lower (2-4%) than at 130 ng l(-1) 4-nonylphenol and in the control. At 4-nonylphenol concentrations of 750 ng l(-1) only 23.8 +/- 1.2% of the larvae survived to the end of the yolk sac stage, at 280 ng l(-1) 53.7 +/- 8.2%, at 130 ng l(-1) 73.8 +/- 1.5%, and in the control 70.9 +/- 1.8%. Sperm motility was not affected by 4-nonylphenol as sperm motility rate, swimming velocity, swimming pattern and motility duration were similar in water and in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol. Incubation of eggs in physiological saline solution containing of 100, 250, or 750 ng l(-1) 4-nonylphenol did not change their fertilizability in comparison to the control. Therefore, 4-nonylphenol did not affect the egg viability. Also the fertilization process (sperm egg contact) was not influenced by 4-nonylphenol as the fertilization rate (percentage of hatched larvae) was similar to the control when eggs were fertilized in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol.     

Bioavailability of Cd to the common carp, Cyprinus carpio, in the presence of humic acid

The uptake of cadmium by the common carp, Cyprinus carpio, was studied in chemically defined freshwater in the absence and presence of commercial humic acid. This was done to evaluate whether the cadmium uptake by carp in the presence of humic acid was related to the ambient Cd2+ -ion activity or whether the complexed metal also contributed to the uptake. Uptake of Cd during a 3-h period of exposure was used as a measure of the biological availability of the metal. The uptake rate data for Cd in total fish and gills obtained in the absence (control) and presence (treatment) of humic acid were analyzed using a Michaelis-Menten model for mediated transport. The Michaelis-Menten parameters KM and Vmax obtained in the control and the treatment experiment were compared for each of the two investigated carp compartments (total fish and gills). The model parameter estimates for Cd uptake by total carp in the treatment experiment (KM = 0.41 +/- 0.11 micromol l(-1); Vmax = 0.66 +/- 0.13 micromol kg(-1) h(-1)) were not significantly different from the model parameters in the control experiment (KM = 0.34+/-0.06 micromol l(-1); Vmax = 0.58+/-0.07 micromol kg(-1) h(-1)) on the basis of Welch's approximate t-test. Similarly, the Michaelis-Menten model parameter estimates for Cd uptake by carp gills in the treatment experiment (KM = 0.15 +/- 0.06 micromol l(-1); Vmax = 5.14 +/- 1.07 micromol kg(-1) h(-1)) were not significantly different from the model parameters derived from the control experiment (KM =0.27 +/- 0.09 micromol l(-1); Vmax = 7.63+/-1.38 micromol kg(-1) h(-1)). This indicated that the Cd uptake rate by total carp and in carp gills in the presence of commercial humic acid followed the measured variations in Cd2+ -ion activity as predicted by the control experiment.     

Food colorant Sunset Yellow (E110) intervenes developmental profile of zebrafish (Danio rerio)

In this study, we tested the teratogenic/embryotoxic potentials of food colorant, Sunset Yellow (E110) using zebrafish embryos as a model. Laboratory‐raised developing embryos of Danio rerio were exposed to graded concentrations (00, 0.1, 1.0, 2.0, 3.0, 4.0, 5.0, 10, 20, 30, 40, 50 and 100 mm ) of E110 from gastrulation stage (~6 hours post‐fertilization [hpf]) up until hatching. The developmental trajectory of each embryo and post‐hatched larva was traced from 24 to 168 hpf. The no observed effect concentration (NOEC), median effective concentration (EC₅₀), median lethal concentration (LC₅₀) and teratogenic index were determined. In the 0.1 mm E110‐exposed embryos, the development proceeded as in controls (NOEC), while, exposure of embryos to 1‐5 mm of E110 led to a decrease in body size, dry body mass of resultant larvae along with appearance of morphological deformities such as, microphthalmia, pericardial edema, yolk sac edema and spinal curvature. Larvae of 10‐50 mm E110‐exposed embryos exhibited increased cellular apoptosis in the cardiac region with significantly declined heartbeats and elevated mortality rates, in addition to the above‐mentioned abnormalities. In the 100 mm exposure group, all embryos succumbed to death within 24 hpf. The NOEC and LC₅₀ recorded were at 0.1 and 42.57 mm respectively. EC₅₀ (96 hpf) recorded for pericardial edema and yolk sac edema was 19.41 and 39.84 mm with teratogenic index quotient 2.1 and 1.06 respectively The study provides direct evidence for the developmental toxicity/teratogenic potential of E110.     

Developmental lead acetate exposure induces embryonic toxicity and memory deficit in adult zebrafish

Lead is a persistent metal and commonly present in our living environment. The present study was aimed to investigate lead-induced embryonic toxicity, behavioral responses, and adult learning/memory deficit in zebrafish. Lead acetate (PbAc) induced malformations such as uninflated swim bladder, bent spine and yolk-sac edema with an EC₅₀ of 0.29 mg/L at 120 h post fertilization (hpf). Spontaneous movement as characterized by tail bend frequency was significantly altered in zebrafish embryos following exposure to PbAc. Behavior assessment demonstrated that lead exposure changed behavioral responses in zebrafish larvae, as hyperactivity was detected within the first minute of light-to-dark transition in the fish exposed to PbAc from 6 to 96 hpf, and a different dose-dependent change was found in swimming speeds in the dark and in the light at 120 hpf following lead exposure. Learning/memory task assay showed that embryos exposed to PbAc from 6 to 120 hpf developed learning/memory deficit at adulthood as exhibited by a significant decrease in accuracy rate to find the food and a significant increase in finding time. Overall, our results suggested that low dose of developmental lead exposure resulted in embryonic toxicity, behavioral alteration, and adult learning/memory deficit in zebrafish.     

β-氯氰菊酯对斑马鱼胚胎的发育毒性

目的以斑马鱼胚胎为模型,探讨一种高效氯氰菊酯β-氯氰菊酯对胚胎发育的影响。方法 丙酮为助溶剂,配制β-氯氰菊酯0.05,0.1,0.15,0.2,0.6和1 mg.L-1,采用换水式每12 h更换一半β-氯氰菊酯溶液,对斑马鱼胚胎进行96 h暴露处理,采用显微镜观察β-氯氰菊酯0.05,0.1,0.15,0.2,0.6和1 mg.L-1对斑马鱼胚胎发育形态,测定受精后24 h(24 hpf)自主抽动次数、48 hpf心率及孵化率、72和96 hpf体轴弯曲个体比例等。结果 与正常对照组比较,β-氯氰菊酯0.05,0.1,0.15,0.2,0.6和1 mg.L-1组斑马鱼胚胎在24 hpf前形态上未出现明显异常,48 hpf以后表现出体轴弯曲、心包囊肿等不同程度的毒性反应症状,β-氯氰菊酯0.2 mg.L-1组幼鱼胸鳍发育即受到严重抑制且黑色素减少体色偏黄;随着β-氯氰菊酯浓度的增加,斑马鱼胚胎在24 hpf时每分钟自主抽动次数由正常对照组的(0.72±0.19)次增加至(3.83±1.07)次(P<0.05);48 hpf孵化率由对照组的(15.5±4.3)%升高至(98.9±1.2)%(P<0.05)。β-氯氰菊酯0.05 mg.L-1组72 hpf和96 hpf体轴弯曲个体比例分别为6.6%和10%,β-氯氰菊酯1 mg.L-1组分别为97.8%和100%。结论 β-氯氰菊酯对斑马鱼胚胎的神经及形态发育均有明显抑制作用,并且呈现一定的时间剂量依赖性。     

Exposure to glibenclamide increases rat beta cells sensitivity to glucose

An increased sensitivity to glucose was observed in islets pre-exposed for 1 h to glibenclamide (0.1 micromol 1(-1)), but not to tolbutamide (100 micromol l(-1)), as indicated by a shift to the left of the dose-response curve (EC(50) at 5.8+/-0.3 mmol l(-1) glucose vs 10.6+/-0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l(-1) glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion-enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124+/-7 vs 51+/-9 nmol microgram prot(-1) 90 min(-1), P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l(-1) dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC(50) 10.9+/-1.3 mmol l(-1) glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial-bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.     

Morphological abnormalities and sensorimotor deficits in larval fish exposed to dissolved saxitoxin

The dietary uptake of one suite of dinoflagellate-produced neurotoxins, that are commonly called paralytic shellfish poisoning (PSP) toxins, is known to cause acute fish kills. However, little is known about the effects of dissolved phase exposure and the potential sublethal effects of this route of exposure on early developmental stages of fish. Toxin exposure during early development is of particular concern because the embryos and larvae of some marine fish species may be unable to actively avoid the dissolved toxins that algal cells release into the water column during harmful algal blooms. Here we use the zebrafish (Danio rerio) as a model experimental system to explore the sublethal effects of a dissolved PSP toxin, saxitoxin (STX), on early development in fish, including sensorimotor function, morphology, and long-term growth and survival. Aqueous phase exposures of 229 +/- 7 microg STX eq. l(-1) caused reductions in sensorimotor function as early as 48 h postfertilization (hpf) and paralysis in all larvae by 4 days postfertilization (dpf). Rohon-Beard mechanosensory neurons appeared to be more sensitive to STX than dorsal root ganglion neurons at this dose. Additionally, exposure to 481 +/- 40 microg STX eq. l(-1) resulted in severe edema of the eye, pericardium, and yolk sac in all exposed larvae by 6 dpf. The onset of paralysis in STX-exposed larvae was stage-specific, with older larvae becoming paralyzed more quickly than younger larvae (5 h at 6 dpf as compared to 8 and 46 h for 4 and 2 dpf larvae, respectively). When transferred to clean water, many larvae recovered from the morphological and sensorimotor effects of STX. Thus, the sublethal effects of the toxin on larval morphology and behavior were reversible. However, zebrafish exposed to STX transiently during larval development (from 2 to 4 dpf) had significantly reduced growth and survival at 18 and 30 days of age. Collectively, these data show that (1) dissolved phase STX is bioavailable to fish embryos and larvae, (2) the toxin is a paralytic with potencies that are stage-specific for fish larvae, (3) the observed toxicological effects of STX exposure are reversible, and (4) a short-term toxin exposure can negatively impact the survival of fish several weeks later. Dissolved algal toxins may therefore have important sublethal effects on vulnerable species of fish.     

多柔比星斑马鱼胚胎心脏发育毒性表现

目的通过观察多柔比星的斑马鱼胚胎心脏毒性表现,为其作为心脏毒性评价模型提供可靠的检测指标。方法选择发育正常的6hpf(hours post fertilization)斑马鱼胚胎暴露于多柔比星2.16～34.48μmol·L-148h。显微镜观察72hpf斑马鱼心血管系统的形态学改变。通过DVC摄像系统测定心率;测量静脉窦-动脉球(SV-BA)间距,HE染色观察斑马鱼心肌结构。结果多柔比星2.16μmol·L-1组斑马鱼心血管系统形态无改变,但心率下降,为(166±5)min-1;SV-BA间距增加,为(237±13)μm。多柔比星4.31μmol·L-1可引起斑马鱼出现心包水肿,心脏畸形,心率减低,为(166±5)min-1;SV-BA间距增加,为(268±13)μm。随着多柔比星浓度增加,斑马鱼出现体长缩短、体节发育异常、脊柱弯曲、卵黄囊水肿、出血、心脏缩小等多种表型改变。心脏组织切片显示,多柔比星8.62μmol·L-1可引起斑马鱼心包腔变大、心脏缩小、心肌层变薄和心肌细胞减少。结论多柔比星对斑马鱼胚胎心脏毒性作用的表现与对哺乳动物的毒性作用表现相同,有望成为评价药物心脏发育毒性的模型。     

Neural defects and cardiac arrhythmia in fish larvae following embryonic exposure to 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47)

Polybrominated diphenyl ethers (PBDEs) are added to plastics, polyurethane foam, and textiles as a flame retardant. While PBDEs play a key role in reducing loss of human life and property from fires, these flame retardants have become pervasive organic contaminants in the environment and in the tissues of fish, birds, marine mammals, and humans. Levels of PBDEs in wildlife and humans continue to rise, raising concerns about potential ecological and health risks associated with exposure to these chemicals. Nevertheless, there is little currently known about the toxicological effects of PBDE exposure. Here, we examined the developmental toxicity of the PBDE congener 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) using the zebrafish (Danio rerio) as an ontogenetic model. Zebrafish embryos were exposed continuously to dissolved phase PBDE 47 (100-5000 microg/l) beginning 3-5 h post-fertilization (hpf). Fish treated with the highest concentrations of PBDE 47 delayed hatching, had reduced growth post-hatching, and displayed an abnormal dorsal curvature of the body with flexion at the hindbrain. By 96h post-fertilization larvae exposed to PBDE 47 had significant tachycardia, which progressed into atrioventricular block arrhythmias. Microinjection of fluorescent dye into the hindbrain ventricle revealed that cerebrospinal fluid in the neural tube and brain ventricles flowed more slowly in fish larvae exposed to PBDE 47, a likely etiology for the dorsal curvature. Similar, though much less pronounced, developmental toxicity also occurred in larvae exposed to PBDE 47 only for a 20h period during early embryogenesis (3-23 hpf), suggesting that PBDEs incorporated in lipid of the egg are bioavailable and cause toxicity later in life. Taken together, this work indicates that exposure to PBDE 47 can cause morphological abnormalities, impair cardiovascular function and cerebrospinal fluid flow, and provides a tractable starting point for using the zebrafish model to explore molecular mechanisms of PBDE toxicity.     

The effect of ketoconazole on adrenal and testicular steroidogenesis in vitro

The effect of the anti-fungal agent, ketoconazole, on cortisol secretion from adrenal cells stimulated with ACTH (1-24, 50 ng/l) and on testosterone secretion from Leydig cells stimulated with LH (5 i.u./l), has been tested. The concentration of drug which inhibited cortisol and testosterone secretion by 50% (ED50) was 3.6 +/- 0.7 mumol/l and 0.61 +/- 0.03 mumol/l, respectively. The effect of ketoconazole on adrenal and testicular steroidogenesis was completely reversible. Thus, adrenal and testicular cells which had been washed after exposure to greater than 95% inhibitory dose of ketoconazole responded in a similar manner to hormone stimulation as cells similarly washed and which had not been exposed to drug. The sites of the anti-steroidogenic effect of ketoconazole have been established using a method based upon the sequential stimulation by the exogenous precursor steroids of the various steps leading to the biosynthesis of cortisol by adrenal cells and testosterone by Leydig cells. We conclude that ketoconazole reversibly inhibits the sequence between ACTH/LH binding and pregnenolone production and also inhibits testicular C-17-C-20, lyase activity.     

Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure

Adult grass shrimp (Palaemonetes pugio) were exposed to endosulfan or methoprene for 96 h and LC(50) values were calculated. Male and female P. pugio cohorts were also exposed to endosulfan for 96 h in an attempt to determine potential differences in sensitivity between the sexes. Results from the methoprene exposure indicated that this pesticide was not acutely toxic to adult grass shrimp at 1 mg l(-1). Due to the lack of sensitivity, sex specific tests with methoprene were not performed. The calculated LC(50) for a population of grass shrimp, including both males and females exposed to endosulfan, was 0.62 microg l(-1). The LC(50) determinations for the sex specific tests were 0.92 microg l(-1) for males and 1.99 microg l(-1) for females. Following these acute exposures, reproductively active grass shrimp were chronically exposed to 200 ng l(-1) endosulfan or 1 mg l(-1) methoprene and were allowed to produce embryos. The resulting embryos were assessed for potential sublethal toxicity. There were no observed differences in the percent successfully hatching or larval mortality 3-days post hatch among the treatments. However, endosulfan treated embryos had a significantly increased hatching time (9.76 days compared to 8.72 days in controls). Methoprene treated embryos also took longer to hatch (9.55 days), but this delay was not significantly different from controls. These findings suggest that endosulfan may preferentially affect male grass shrimp and exposed female grass shrimp may produce embryos with delayed hatching times.     

In vitro physiological evidence of enhanced antioxidant and neuroprotective action of 2,3-dihydromelatonin, a melatonin analogue.

As the capacity of the endogenous antioxidative system is limited, pharmacological treatment with antioxidants may help to protect neuronal tissue against increased amount of reactive oxygen species produced during oxidative stress. We attempted to improve resistance of rat hippocampal slices exposed to ischaemia in vitro (hypoxia (HYP) accompanied with decreased glucose concentration followed by reoxygenation (ROX)) and thus to diminish the impairment of synaptic transmission after HYP/ROX. We compared the protective features of the melatonin analogue 2,3-dihydromelatonin (2,3-DHM) with melatonin itself. In preliminary experiments, the compound 2,3-DHM compared to melatonin revealed enhanced antilipoperoxidation action in rat brain homogenates exposed to Fe/ascorbate system (-logIC(50) = 4.76 +/- 0.01 versus -logIC(50) = 2.51 +/- 0.02, respectively). In this study, 2,3-DHM (from 0.3 to 10 micromol l(-1)) applied at 30 min before the beginning of HYP and remaining all over the 6-min HYP as well as 20-min ROX, exerted a protective effect demonstrated by improvement of the population spike amplitude (PoS) recovery during ROX, with the maximum effect at 3 micromol l(-1). In accordance with this, the ratio of irreversibly damaged slices after HYP/ROX was decreased in the groups treated with 2,3-DHM. Moreover, a significant delay of PoS decay during HYP (expressed as half time, t(0.5)) was revealed at 2,3-DHM concentration 1 and 3 micromol l(-1)). An equipotent effect of melatonin and 2,3-DHM was achieved by a 100-times lower concentration of the latter (0.3 and 1 micromol l(-1)) compared to that of melatonin (30 and 100 micromol l(-1)). Further, compared to the highest effect of 2,3-DHM in the concentration 3 micromol l(-1) on the percentage of irreversibly damaged slices (only 20%), melatonin did not exert such pronounced effect, either in the concentration 30 or 100 micromol l(-1) (67 and 50%, respectively). We conclude that hydrogenation of the melatonin molecule significantly improved its antihypoxic effect in our model of rat hippocampal slices exposed to ischaemic conditions in vitro, similarly as it enhanced the antilipoperoxidation action of 2,3-DHM in our previous studies. These findings suggest that 2,3-DHM deserves more attention concerning its neuroprotective effect in oxidative stress-associated tissue damage.     

Swimming performance and energy metabolism of rainbow trout, common carp and gibel carp respond differently to sublethal copper exposure

We compared the effects of sublethal waterborne copper exposure on swimming performance and respiration rates in rainbow trout, Oncorhynchus mykiss, with those in less sensitive cyprinid species such as common carp, Cyprinus carpio, and gibel carp, Carassius auratus gibelio. These cyprinids are considerably more resistant to Cu intoxication, and differ from trout in swimming performance and respiratory behaviour. Critical swimming speed (U(crit)), oxygen consumption, plasma ammonia and muscle ammonia, lactate and pH were measured during a 28-day sublethal exposure to 1 microM Cu. U(crit) decreased with 48, 31 and 13% within the first 12-24 h for rainbow trout, common and gibel respectively. Gibel carp recovered quickly and experienced no further reduction in swimming performance. Recovery of swimming capacity in rainbow trout and common carp was only partial. All three species displayed similar plasma ammonia peaks in the first hours to days, and a more gradual muscle ammonia accumulation over time. Whereas no signs of respiratory stress occurred in rainbow trout, common carp experienced a transient reduction in oxygen consumption combined with anaerobic metabolism after 24 h of exposure. At the same time, oxygen consumption was also reduced in gibel carp, but no signs of anaerobic metabolism were detected. Cu accumulated quickly to similar levels (36-39 microg g(-1) dry weight at day 3) in the gills of all three species, after which accumulation leveled off. Liver tissue of rainbow trout had a high Cu level from the start, and Cu concentration did not show any additional accumulation. In contrast, common carp liver showed a significant Cu accumulation from day 3 onwards, while accumulation in gibel livers was much slower and was significant from day 7 onwards. Interestingly, Cu accumulation patterns in plasma and kidney revealed a possibly important role for the kidney in Cu homeostasis of gibel carp.