W. De Coen

Effect of dietary non-protein energy levels on condition and oxidative status of Senegalese sole (Solea senegalensis) juveniles

The effects of dietary non-protein energy levels on growth, oxidative status and condition were studied in juveniles of Senegalese sole (Solea senegalensis). Four isonitrogenous diets with four energy levels were used: A-low (11% lipid, raw carbohydrate); B-intermediate (11% lipid, digestible carbohydrate); C-intermediate (21% lipid, raw carbohydrate); and D-high (21% lipid, digestible carbohydrate). Survival, relative growth rate (RGR) and feed conversion rate were not significantly affected by the dietary treatments. The fatty acid composition of the fish muscle varied little among the treatments, but trans 18:2n - 6, total n - 6 and EPA/DHA ratio, tended to be higher in fish fed diets with low lipid level. Cellular energy allocation (CEA) results (indicative of metabolic status and net energy budgets) showed significant differences in liver, but not in muscle samples. Livers of fish fed diet C contained the lowest carbohydrate, protein and CEA values, but the highest cellular energy consumption. Fish fed diet A had the highest CEA for growth, followed by fish fed diets B and D and then diet C. The liver and muscle peroxidation and antioxidant activity were measured by thiobarbituric acid reactive substances (TBARS) test and the enzymatic activities levels of catalase (CAT) and superoxide dismutase (SOD). TBARS values were higher for fish fed diets with high lipid content. However, no clear relation was found between HUFA level and TBARS value. The activity levels of the antioxidant enzymes CAT and SOD were higher in livers of fish fed diets with a high lipid level. Furthermore, CAT and SOD activity and TBARS values were influenced by the type of dietary starch in the diet. Higher oxidation rates were observed in fish fed diets containing raw carbohydrate. These data suggest that lipid and carbohydrate energy sources affect the oxidative status of Senegalese sole. Diets containing low levels of lipid and digestible starch reduce the susceptibility of the fish to oxidation and may enhance growth rate

Environmental pollutants and type 2 diabetes: a review of mechanisms that can disrupt beta cell function

The prevalence of diabetes mellitus is currently at epidemic proportions and it is estimated that it will increase even further over the next decades. Although genetic predisposition and lifestyle choices are commonly accepted reasons for the occurrence of type 2 diabetes, it has recently been suggested that environmental pollutants are additional risk factors for diabetes development and this review aims to give an overview of the current evidence for this. More specifically, because of the crucial role of pancreatic beta cells in the development and progression of type 2 diabetes, the present work summarises the known effects of several compounds on beta cell function with reference to mechanistic studies that have elucidated how these compounds interfere with the insulin secreting capacity of beta cells. Oestrogenic compounds, organophosphorus compounds, persistent organic pollutants and heavy metals are discussed, and a critical reflection on the relevance of the concentrations used in mechanistic studies relative to the levels found in the human population is given. It is clear that some environmental pollutants affect pancreatic beta cell function, as both epidemiological and experimental research is accumulating. This supports the need to develop a solid and structured platform to fully explore the diabetes-inducing potential of pollutants.

Toxicity evaluation of perfluorooctane sulfonate (PFOS) in the liver of common carp (Cyprinus carpio).

Perfluorooctane sulfonate (PFOS) has been manufactured for over 50 years in increasing quantities and has been used for several industrial and commercial aims. Due to persistence and bioaccumulation of this pollutant, it can be found worldwide in wildlife and humans. Biochemical effects of PFOS exposure are mainly studied in mammalian model species and information about effects on fish species remain largely scarce. This lack of toxicity data points out that there is an urgent need for the mechanistic molecular understanding of the mode of action of this pollutant. In the present study, common carp (Cyprinus carpio) was exposed through water for 14 days at concentrations of 0.1, 0.5 and 1 mg/l PFOS. Liver was selected as target tissue. Custom microarrays were constructed from cDNA libraries obtained with Suppression Subtractive Hybridization-Polymerase Chain Reaction (SSH-PCR) experiments. Microarray data revealed that the expression of several genes in the liver was influenced by PFOS exposure and real-time PCR was used to confirm these gene expression changes. The affected genes were mainly involved in energy metabolism, reproduction and stress response. Furthermore, the relative condition factor, the hepatosomatic index, and the available glycogen reserves of the exposed fish were significantly lower after 14 days of exposure than in the control fish. At all levels of biological organization, indications of a trade-off between the metabolic cost of toxicant exposure on one hand and processes vital to the survival of the organism on the other hand were seen. Our results support the prediction that increases in energy expenditure negatively affects processes vital to the survival of an organism, such as growth.

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.

Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio).

In the present study we evaluated the toxicological effects of a scarcely documented environmental pollutant, perfluorooctane sulfonic acid (PFOS), on selected biochemical endpoints in the common carp, Cyprinus carpio. Juvenile organisms were exposed to PFOS through a single intraperitoneal injection (liver concentrations ranging from 16 to 864 ng/g after 5 days of exposure) and after 1 and 5 days effects were assessed in liver and serum of the exposed organisms. The investigation of the hepatotoxicity of PFOS included the determination of the peroxisome proliferating potential (peroxisomal palmitoyl CoA oxidase and catalase activity) and the compounds influence on the average DNA basepair length (ABPL) by agarose gel electrophoresis. Total antioxidant activity (TAA), cholesterol and triglyceride levels were monitored in the serum. After 1 day of exposure the ABPL was significantly increased in the 270 and 864 ng/g treatment groups. After 5 days of exposure significant increases relative to the control were observed for the 16, 270 and 864 ng/g treatment groups. Enzyme leakage from the liver was investigated by measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum. At 561, 670 and 864 ng/g PFOS a significant increase in serum ALT activity became apparent after 5 days of exposure with values ranging from 159 to 407% relative to the control. For serum AST activity a significant increase for the 864 ng/g treatment group was observed with a value of 112% relative to the control. Determination of the polymorphonuclear leukocyte migration into liver tissue as assessed through myeloperoxidase (MPO) activity in liver, was used as an indicator for inflammation. It appeared that inflammation was not involved in the observed membranous enzyme leakage for the 561, 670 and 864 ng/g PFOS treatment groups. The results of this study suggest that PFOS induces inflammation-independent enzyme leakage through liver cell membranes that might be related to cell necrosis. Furthermore, results show that PFOS does not significantly affects serum antioxidant levels nor does it clearly induce peroxisome proliferation in carp. This study also points out that PFOS might interfere with homeostasis of the DNA metabolism. The results of these biochemical analyses were used to perform an initial hazard assessment study indicating that PFOS levels observed in tissues of wildlife populations could induce a clear rise in serum transaminase levels indicative for disruption of hepatocyte membrane integrity.

Hypoxia/Ischemia and the Regulation of Neuroglobin and Cytoglobin Expression

In analogy to hemoglobin (Hb) and myoglobin (Mb), neuroglobin (Ngb) and cytoglobin (Cygb) are supposed to be involved in oxygen (O2) storage and delivery. The Cygb gene harbours both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen‐dependent regulation. We examined the relative transcriptional changes of Ngb and Cygb in a situation of chronic hypoxia using real‐time quantitative PCR. We could conclude that Cygb is a hypoxia‐induced gene, which is transcriptionally upregulated during chronic hypoxia in a hippocampal neuronal cell line and in multiple murine metabolically active tissues. The mechanism of induction of Cygb is HIF‐1α dependent. HIF‐1 is unique among mammalian transcription factors with respect to the specificity and sensitivity of its induction by hypoxia. Ngb expression seems to be regulated using other response elements and is less influenced by hypoxia. IUBMB Life, 56: 681‐687, 2004

The search for alternative aqueous film forming foams (AFFF) with a low environmental impact: physiological and transcriptomic effects of two Forafac(®) fluorosurfactants in turbot.

Fluorosurfactants are the key components in aqueous film forming foams (AFFF). They provide these fire fighting agents with the required low surface tension and they enable film formation on top of lighter fuels to prevent burn back. Development of effective and environmentally acceptable PFOS alternatives is one of the most important priorities in the fire fighting foam industry. DuPont™ offers the fluorosurfactant mixtures Forafac(®)1157 and Forafac(®)1157N for the formulation of AFFFs which are alternatives to the persistent and toxic perfluorooctane sulphonate (PFOS). Ecotoxicological testing of these inadequately documented mixtures is necessary to include them in AFFF hazard and risk assessment. Juvenile turbot (Scophthalmus maximus) were exposed for 14 days to 0.1; 0.5 and 1.5mg/L of the fluorosurfactant mixtures used in Forafac(®)1157 and Forafac(®)1157N. In an initial transcriptomics experiment, microarray analysis revealed differentially expressed transcripts of genes which were mainly involved in digestion and in the immune system. This discovery-driven screening approach offered the basis for new hypotheses that were tested in two subsequent experiments in which food intake, energy reserves, growth and a set of haematological parameters were examined. Additionally, effects of the two mixtures were compared to those of PFOS. Based on the results of this study, the mode of action of Forafac(®)1157N was the activation of the acute phase reaction resulting in increased leukocyte concentrations and the inhibition of growth due to the high energetic cost of toxicant exposure. For Forafac(®)1157, evidences of immunosuppression were found on the transcriptional level and the altered differential leukocyte profiles indicated that stress was induced in these fish. However, food intake, energy reserves and growth were not compromised, even at high exposure concentrations, which was in contrast to the effects seen after PFOS exposure. Taking into account that Forafac(®)1157 appeared to be less toxic than PFOS, this mixture could be considered as a more environmentally acceptable PFOS alternative for the use in AFFFs.

Intersex and sterility in the periwinkle Littorina littorea (Mollusca: Gastropoda) along the Western Scheldt estuary, The Netherlands.

In this study we present the results of an intersex survey of Littorina littorea along the heavily polluted Western Scheldt estuary (the Netherlands), and record for the first time the intersex phenomenon in L. littorea from Dutch waters. Intersex differed significantly between localities and was the highest in the vicinity of the harbours of Antwerp and Vlissingen, as reflected by the I

Integration of molecular with higher-level effects of dietary zinc exposure in Daphnia magna.

We exposed Daphnia magna for 21 days to dietary Zn, incorporated in a diet of the green alga Pseudokirchneriella subcapitata at 720 microg Zn/g dry wt and compared its response to D. magna fed with a control diet (116 microg Zn/g dry wt). Exposure to dietary Zn resulted in an increased body burden of D. magna (93.7 microg/g dry wt vs. 61.3 microg/g dry wt in the control) but did not affect survival, growth, or feeding rate. Only reproduction was significantly reduced from the 2nd brood onwards. Gene expression analysis, using microarray analysis and RT-PCR, showed that dietary Zn exposure resulted in the differential expression of several genes involved in molting-associated processes (i.e., chitin binding, cuticle metabolism), especially after 6 days of exposures (but not after 13 or 21 days of exposure). Monitoring of time to molt and intermolt-period confirmed this molting effect at the organism level in the first week of exposure. The data suggest a possible link between Zn-induced effects on molting-related processes and reproductive inhibition, but this link is only obvious for effects on the 2nd brood size and not for later broods. Reproductive inhibition in later broods may also be explained by a disturbed mitochondrial function, but more research is clearly needed to give a more definitive integrated explanation of the observed effects at the molecular and organism level.

Dose–response relationships and statistical performance of a battery of bacterial gene profiling assays

Because of increasing awareness and legislative demands, there is a demand for the development and use of biological assays for the assessment of the toxicity of chemicals, environmental samples. Recently, a growing number of bacterial reporter assays have been developed and implemented. Nevertheless, little data is published on the performance of these assays in terms of analytical parameters. We present results on a battery of 14 transgenic Escherichia coli strains carrying different promoter::reporter fusions. Growth characteristics and basal expression levels were modeled and fitted, data show that growth curves should be taken into account during test development. Our study shows that the induction profiles reflect the mode of action, e.g., paraquat clearly induces the soxRS operon. The sensitivity of the assay compares well to that of whole organism tests, e.g., fish and Daphnia for polar organics. Metal toxicity is detected less efficiently, e.g., cadmium is detected near the LC50 of carp, considered a relatively insensitive species towards cadmium. The assay variability ranges from 10 to 40% depending on the strain, comparable to that of other bioassays. The variability was shown to be determined by the intrinsic traits of the promoter–strain combination, not by operating conditions.