An ecotoxicological view on neurotoxicity assessment.

The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.

Microarray-based annotation of the gut transcriptome of the migratory locust, Locusta migratoria.

The migratory locust, Locusta migratoria, is a serious agricultural pest and important insect model in the study of insect digestion and feeding behaviour. The gut is one of the primary interfaces between the insect and its environment. Nevertheless, knowledge on the gut transcriptome of L. migratoria is still very limited. Here, 48 802 expressed sequence tags were extracted from publicly available databases and their expression in larval gut and/or brain tissue was determined using microarray hybridization. Our data show 2765 transcripts predominantly or exclusively expressed in the gut. Many transcripts had putative functions closely related to the physiological functions of the gut as a muscular digestive organ and as the first barrier against microorganisms and a wide range of toxins. By means of a ranking procedure based on the relative signal intensity, we estimated 15% of the transcripts to show high expression levels, the highest belonging to diverse digestive enzymes and muscle-related proteins. We also found evidence for very high expression of an allergen protein, which could have important implications, as locusts form a traditional food source in various parts of the world, and were also recently added to the list of insects fit for human consumption in Europe. Interestingly, many highly expressed sequences have as yet unknown functions. Taken together, the present data provide significant insight into locust larval gut physiology, and will be valuable for future studies on the insect gut.

PFOS affects posterior swim bladder chamber inflation and swimming performance of zebrafish larvae.

Perfluorooctane sulphonate (PFOS) is one of the most commonly detected perfluorinated alkylated substances in the aquatic environment due to its persistence and the degradation of less stable compounds to PFOS. PFOS is known to cause developmental effects in fish. The main effect of PFOS in zebrafish larvae is an uninflated swim bladder. As no previous studies have focused on the effect of PFOS on zebrafish swim bladder inflation, the exact mechanisms leading to this effect are currently unknown. The objective of this study was to determine the exposure windows during early zebrafish development that are sensitive to PFOS exposure and result in impaired swim bladder inflation in order to specify the mechanisms by which this effect might be caused. Seven different time windows of exposure (1-48, 1-72, 1-120, 1-144, 48-144, 72-144, 120-144h post fertilization (hpf)) were tested based on the different developmental stages of the swim bladder. These seven time windows were tested for four concentrations corresponding to the EC-values of 1, 10, 80 and 95% impaired swim bladder inflation (EC1=0.70 mg L(-1), EC10=1.14 mg L(-1), EC80=3.07 mg L(-1) and EC95=4.28 mg L(-1)). At 6 days post fertilization, effects on survival, hatching, swim bladder inflation and size, larval length and swimming performance were assessed. For 0.70 mg L(-1), no significant effects were found for the tested parameters while 1.14 mg L(-1) resulted in a reduction of larval length. For 3.07 and 4.28 mg L(-1), the number of larvae affected and the severity of effects caused by PFOS were dependent on the time window of exposure. Exposure for 3 days or more resulted in significant reductions of swim bladder size, larval length and swimming speed with increasing severity of effects when the duration of exposure was longer, suggesting a possible effect of accumulated dose. Larvae that were only exposed early (1-48 hpf) or late (120-144 hpf) during development showed no effects on the studied endpoints. The results demonstrate that PFOS does not affect the budding phase, and does not cause deflation of already inflated swim bladders. PFOS clearly affects processes that take place during the inflation phase and might also have an effect on the formation of the tissue layers forming the swim bladder.

Mechanistic toxicity study of perfluorooctanoic acid in zebrafish suggests mitochondrial dysfunction to play a key role in PFOA toxicity.

The aquatic environment is an important site for perfluorooctanoic acid (PFOA) deposit. Nevertheless, the exact mode of action and its resulting toxicological effects in aquatic organisms remain largely unknown. To gain a better understanding of the mode of action of teleost PFOA toxicity, transcriptomics, proteomics, biochemical parameters and reproduction were integrated in this study. Male and female zebrafish were exposed to nominal concentrations of 0.1, 0.5 and 1 mg L(-1) PFOA for 4 and 28 d resulting in PFOA accumulation which was higher in males than in females. These gender-related differences were likely caused by different elimination rates due to distinct hormone levels and differences in transport activity by solute carriers. The general mode of action of PFOA was described as an increase of the mitochondrial membrane permeability followed by an impairment of aerobic ATP production. Depletion of liver glycogen stores together with altered expression levels of transcripts involved in carbohydrate metabolism, with emphasis on anaerobic metabolism, was probably a means of compensating for this decreased aerobic efficiency. The mitochondrial dysfunction further resulted in effects on oxidative stress and apoptosis at the gene transcript and protein level. As a consequence, evidence for the replacement of the affected cells and organelles to sustain tissue homeostasis was found at the transcript level, resulting in an even greater glycogen depletion. Despite this increase in metabolic expenditure, no effects on reproduction were found indicating that the fish seemed to cope with exposure to the tested concentrations of PFOA during the exposure period of 1 month.

Structure-activity relationship assessment of four perfluorinated chemicals using a prolonged zebrafish early life stage test.

Perfluorinated compounds (PFCs) are a group of anthropogenic chemicals containing diverse functional groups and chain lengths. They are known to be persistent and bioaccumulative explaining their worldwide environmental presence. The toxicological information on these chemicals is still incomplete and insufficient to assess their environmental impact and structure-activity relationship. In the present study, the developmental effects of PFOS (perfluorooctane sulfonate, C8), PFOA (perfluorooctanoic acid, C8), PFBS (perfluorobutane sulfonate, C4) and PFBA (perfluorobutanoic acid, C4) were evaluated in zebrafish embryos (Danio rerio). The different chain lengths and functional groups of the selected chemicals made it possible to determine the structure-activity relationship of these compounds. PFCs with longer chain lengths (C8) tend to be more toxic than PFCs with shorter chain lengths (C4). Comparison based on the functional groups of compounds with the same chain length indicates that PFCs with a sulfonate group have a larger toxic potential than the ones with a carboxyl group. Furthermore, exposure to the different PFCs resulted in some general effects, such as deformations of the tail and an uninflated swim bladder, as well as in more specific effects which might be related to the structure of the tested chemicals. Oedemas and effects on length could only be detected in 8-carbon PFCs while malformations of the head were a more specific action of the sulfonated PFCs. Effects on hatching rate and success were found in PFOA exposed embryos and heart rates were affected after exposure to PFOS, PFOA and PFBS.

Creatine loading does not impact on stroke performance in tennis.

The effect of acute creatine supplementation on stroke quality was investigated during simulated match play. Well-trained tennis players reported to the test center on two occasions. On each occasion they performed the Leuven Tennis Performance Test (LTPT) and a 70 m shuttle run (SHR). During 5 days prior to each test session they received in random order and according to a double-blind cross-over study design either oral creatine supplements (4 x 5 g per day) or placebo. The two experimental periods were separated by a 5-week washout period. Stroke quality was evaluated during the LTPT by means of registration of error rate and measurement of ball velocity and precision of lateral and longitudinal ball placement. Compared with placebo, creatine supplementation did not significantly impact on either power or precision of first and second services, baseline strokes in neutral and defensive rallies, and volleys. Shuttle run time was 19.87 +/- 0.30 sec during placebo versus 19.85 +/- 0.27 sec during creatine treatment. Acute creatine supplementation does not enhance stroke performance or sprint power in match-like conditions in elite tennis players.

Evaluation of stroke performance in tennis.

In the present studies, the Leuven Tennis Performance Test (LTPT), a newly developed test procedure to measure stroke performance in match-like conditions in elite tennis players, was evaluated as to its value for research purposes. The LTPT is enacted on a regular tennis court. It consists of first and second services, and of returning balls projected by a machine to target zones indicated by a lighted sign. Neutral, defensive, and offensive tactical situations are elicited by appropriately programming the machine. Stroke quality is determined from simultaneous measurements of error rate, ball velocity, and precision of ball placement. A velocity/precision (VP) an a velocity/precision/error (VPE) index are also calculated. The validity and sensitivity of the LTPT were determined by verifying whether LTPT scores reflect minor differences in tennis ranking on the one hand and the effects of fatigue on the other hand. Compared with lower ranked players, higher ones made fewer errors (P < 0.05). In addition, stroke velocity was higher (P < 0.05), and lateral stroke precision, VP, and VPE scores were better (P < 0.05) in the latter. Furthermore, fatigue induced by a prolonged tennis load increased (P < 0.05) error rate and decreased (P < 0.05) stroke velocity and the VP and VPE indices. It is concluded that the LTPT is an accurate, reliable, and valid instrument for the evaluation of stroke quality in high-level tennis players.

Carbohydrate supplementation improves stroke performance in tennis.

The effect of carbohydrate supplementation on stroke quality during prolonged simulated tennis match-play was investigated. Well-trained tennis palyers reported to the test center three times. At each occasion they performed a pretest, consisting of the leuven Tennis Performance Test (LTPT) and a shuttle run (SHR), which they repeated (posttest) after a 2-h strenuous training session. Throughout the test session, they received in a double blind random order either a placebo drink (P), a carbohydrate solution (0.7 gxkg(-1) BWxh(-1); CHO), or CHO plus a dose of caffeine (5 mg per kg BW). Stroke quality was evaluated during the LTPT by means of measurements of error rate, ball velocity, precision of ball placement, and a velocity-precision (VP) and a velocity-precision-error (VPE) index. Pretest scores were similar during P and CHO. During P, compared with the pretest, stroke quality during the posttest deteriorated (P < 0.05) both for the first service and strokes during defensive rallies and for SHR performance. However, compared with P, the increase in error rate and number of nonreached balls indefensive rallies was smaller (P < 0.05) during CHO. Similarily, CHO attenuated (P < 0.05) the increase in error rate and the decrease in both the VP (P < 0.1) and VPE (P < 0.05) indices for the first service upon fatigue. Furthermore, CHO improved posttest SHR performance. Stroke quality and SHR time were similar during CHO alone and during combined CHO plus caffeine administration, both for the pretest and for the pretest and for the posttest. It is concluded that CHO supplementation improves stroke quality during the final stages of prolonged tennis play. The data prove that CHO intake may facilitate the maintenance of physical quality during long-lasting intermittent exercise to fatigue.

Adenosine exerts a glycogen-sparing action in contracting rat skeletal muscle.

The role of adenosine in regulating glycogen breakdown during electrically induced muscle contractions was investigated in isolated rat hindquarters perfused with a standard medium either lacking or containing 100 microU/ml insulin and/or 1.67 nM isoprenaline. Nonselective A1/A2-adenosine receptor antagonism via caffeine enhanced (P < 0.05) glycogen breakdown in contracting fast-oxidative (FO) fibers by 40%, provided they were exposed to both insulin and isoprenaline. Combined A1/A2-receptor antagonism by 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) plus 3,7-dimethyl-1-proparglyxanthine (DMPX) fully reproduced (P < 0.05) this stimulatory effect. Furthermore, CPDPX plus DMPX also enhanced (P < 0.05) glycogenolysis during contractions in soleus but not in white gastrocnemius muscle. In contrast, CPDPX or DMPX alone did not affect glycogenolysis in either fiber type. Muscle adenosine 3',5'-cyclic monophosphate concentration during contractions was increased (P < 0.05) by CPDPX plus DMPX in both fiber types, whereas glycogen synthase fractional activity was depressed (P < 0.05). Phosphorylase activity was not changed by CPDPX plus DMPX. It is concluded that adenosine exerts a glycogen-sparing action in oxidative skeletal muscle exposed to both insulin and beta-adrenergic stimulation during contraction, presumably via stimulation of glycogen synthase activity.

Significance of insulin for glucose metabolism in skeletal muscle during contractions.

Glucose uptake rate in active skeletal muscles is markedly increased during exercise. This increase reflects a multifactorial process involving both local and systemic mechanisms that cooperate to stimulate glucose extraction and glucose delivery to the muscle cells. Increased glucose extraction is effected primarily via mechanisms exerted within the muscle cell related to the contractile activity per se. Yet contractions become a more potent stimulus of muscle glucose uptake as the plasma insulin level is increased. In addition, enhanced glucose delivery to muscle, which during exercise is essentially effected via increased blood flow, significantly contributes to stimulate glucose uptake. Again, however, increased glucose delivery appears to be a more potent stimulus of muscle glucose uptake as the circulating insulin level is increased. Furthermore, contractions and elevated flow prove to be additive stimuli of muscle glucose uptake at any plasma insulin level. In conclusion, the extent to which muscle glucose uptake is stimulated during exercise depends on various factors, including 1) the intensity of the contractile activity, 2) the magnitude of the exercise-associated increase in muscle blood flow, and 3) the circulating insulin level.

Important role of insulin and flow in stimulating glucose uptake in contracting skeletal muscle.

The relative role of contractions, insulin, and increased supply of glucose and insulin, via an increase in blood flow, in stimulating glucose uptake in skeletal muscle during contractions was studied in isolated perfused rat hindlimbs. Hindlimbs were perfused with a standard perfusate medium containing 6 mmol/l glucose and four different insulin concentrations (0, 100, 500, and 20,000 microU/ml). Contractions were induced by supramaximal intermittent electrical stimulation of the sciatic nerve. Three different perfusion protocols were used: 1) muscles were stimulated to contract without concomitantly increasing perfusate flow; 2) flow was increased in the absence of electrical stimulation; and 3) muscles were stimulated to contract together with a flow increase. Both contractions and increased flow of perfusate, applied as separate stimuli, increased (P < 0.05) glucose uptake in the absence of insulin. Yet when submaximal insulin concentrations were added to the perfusate, the stimulatory action of both contractions and increased blood flow on muscle glucose uptake was augmented. The higher the submaximal insulin concentration, the greater the increment (P < 0.05). This effect, however, faded at supramaximal insulin concentration. Electrical stimulation associated with an increase in perfusion flow rate produced a greater (P < 0.05) rise in glucose uptake than did contractions alone. In fact, stimulation of muscle glucose uptake by contractions and increased flow proved to be additive at any insulin concentration. We conclude that contractions and increased blood flow act as additional stimuli to muscle glucose uptake at any insulin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine receptors mediate synergistic stimulation of glucose uptake and transport by insulin and by contractions in rat skeletal muscle.

The role of adenosine receptors in the regulation of muscle glucose uptake by insulin and contractions was studied in isolated rat hindquarters that were perfused with a standard medium containing no insulin or a submaximal concentration of 100 microU/ml. Adenosine receptor antagonism was induced by caffeine or 8-cyclopentyl-1,3-dipropylxantine (CPDPX). Glucose uptake and transport were measured before and during 30 min of electrically induced muscle contractions. Caffeine nor CPDPX affected glucose uptake in resting hindquarters. In contrast, the contraction-induced increase in muscle glucose uptake was inhibited by 30-50% by caffeine, as well as by CPDPX, resulting in a 20-25% decrease in the absolute rate of glucose uptake during contractions, compared with control values. This inhibition was independent of the rate of perfusate flow and only occurred in hindquarters perfused with insulin added to the medium. Thus, adenosine receptor antagonism inhibited glucose uptake during simultaneous exposure to insulin and contractions only. Accordingly, caffeine inhibited 3-O-methylglucose uptake during contractions only in oxidative muscle fibers that are characterized by a high sensitivity to insulin. In conclusion, the present data demonstrate A1 receptors to regulate insulin-mediated glucose transport in contracting skeletal muscle. The findings provide evidence that stimulation of sarcolemmic adenosine receptors during contractions is involved in the synergistic stimulation of muscle glucose transport by insulin and by contractions.