Angela Koehler

Effects of nanoparticles in Mytilus edulis gills and hepatopancreas : A new threat to marine life?

Every day new extraordinary properties of nanoparticles (a billionth of a meter) are discovered and worldwide millions are invested into nanotechnology and nanomaterials. Risks to marine organisms are still not fully understood and biomarkers to detect health effects are not implemented, yet. We used the filter feeding blue mussel as a model to analyse uptake and effects of nanoparticles from glass wool, a new absorbent material suggested for use in floating oil spill barriers. In both, gills and hepatopancreas we analysed uptake of nanomaterials by transmission electronmicroscopy (TEM) in unstained ultrathin sections over a period of up to 16 days. Lysosomal stability and lipofuscin content as general indicators of cellular pathology and oxidative stress were also measured. As portals of uptake, diffusion and endocytosis were identified resulting in nanoparticle accumulation in endocytotic vesicles, lysosomes, mitochondria and nuclei. Dramatic decrease of lysosomal membrane stability occurred after 12h of exposure. Lysosomal damage was followed by excessive lipofuscin accumulation indicative of severe oxidative stress. Increased phagocytosis by granulocytes, autophagy and finally apoptosis of epithelial cells of gills and primary and secondary digestive tubules epithelial cells indicated progressive cell death. These pathological responses are regarded as general indices of toxic cell injury and oxidative stress. By the combinational use of biomakers with the ultrastructural localisation of nanoparticle deposition, final evidence of cause-effect relationships is delivered.

Effects of dredging in Göteborg harbor, Sweden, assessed by biomarkers in eelpout (Zoarces viviparus).

We used a battery of biomarkers in fish to study the effects of the extensive dredging in Göteborg harbor situated at the river Göta alv estuary, Sweden. Eelpout (Zoarces viviparus) were sampled along a gradient into Göteborg harbor, both before and during the dredging. Biomarker responses in the eelpout before the dredging already indicated that fish in Göteborg harbor are chronically affected by pollutants under normal conditions compared to those in a reference area. However, the results during the dredging activities clearly show that fish were even more affected by remobilized pollutants. Elevated ethoxyresorufin-O-deethylase activities and cytochrome P4501A levels indicated exposure to polycyclic aromatic hydrocarbons. Elevated metallothionein gene expression indicated an increase in metal exposure. An increase in general cell toxicity, measured as a decrease in lysosomal membrane stability, as well as effects on the immune system also could be observed in eelpout sampled during the dredging. The results also suggest that dredging activities in the Göta alv estuary can affect larger parts of the Swedish western coast than originally anticipated. The present study demonstrates that the application of a set of biomarkers is a useful approach in monitoring the impact of anthropogenic activities on aquatic environments.

Sex-related responses to oxidative stress in primary cultured hepatocytes of European flounder (Platichthys flesus L.)

Effects of oxidative stress induced by xenobiotic compounds were studied in primary cultures of isolated hepatocytes of immature European flounder (Platichthys flesus L.) of both sexes caught in a relatively unpolluted area of the German Bight (North Sea). Cells were exposed to oxidative stressors such as 100 microM hydrogen peroxide (H2O2), 100 microM benzo[a]pyrene (B[a]p) and 50 microM nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-aminohydantoin; NF) for 2 and 24 h. Cell mortality was determined with the use of the fluorescent ethidium homodimer-1 and calcein. Oxidative stress response was assessed by quantitative analysis of (1) intracellular reactive oxygen species (ROS) formation with dihydrorhodamine 123, (2) lipid peroxidation on the basis of concentrations of lipid hydroperoxides and the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and (3) cellular total oxidant-scavenging capacity (TOSC) using the TOSC assay (Winston et al., 1998). An increase in ROS formation was detected as early as 2 h after exposure to H2O2, B[a]p and NF. After 24 h, stress responses were lower, except following exposure to NF. The pattern of responses differed with the different oxidative stressors. Lipid peroxidation and the capacity to scavenge ROS were increased significantly in both sexes only after exposure to H2O2, whereas B[a]p and NF provoked sex-dependent responses. B[a]p-induced lipid peroxidation and increase in scavenging capacity were observed only in hepatocytes of females, whereas NF initiated these responses only in cells of males. Sex differences in oxidative stress response only after exposure to pro-oxidants that require enzymatic activation infer the importance of biotransformation pathways in stress responses. Because of their sensitivity to oxidative stress, flounder hepatocytes provide a useful model for early risk assessment of xenobiotics.

Reduced nicotinamide adenine dinucleotide phosphate and the higher incidence of pollution‐induced liver cancer in female flounder

In biological effect monitoring programs, induction of biotransformation and detoxification enzymes is used as a biomarker for pollution. Yet sex differences are usually neglected in the availability of reduction equivalents needed in these metabolic pathways and may affect biomarker responses. For example, female flounder have a threefold higher incidence of macroscopic liver nodules than males of the same age class in polluted environments of the North Sea that progress toward carcinomas, whereas tumors in males virtually never develop into cancer. Evidence is presented in this review that NADPH plays a significant role in this sex-related response to xenobiotics in liver of flounder. The NADPH is needed for biosynthesis, particularly of lipids and lipoproteins, and detoxification processes such as one-electron and two-electron biotransformation and conjugation and, therefore, its availability as substrate determines biomarker responses. Biotransformation of xenobiotics is more strongly induced and conjugation processes are less affected in male flounder liver during exposure. In female liver, NADPH is required for production of the yolk precursor protein vitellogenin for oocyte production. The latter process has a higher priority than the NADPH-requiring detoxification processes because reproductive success is more relevant in evolutionary perspectives than the survival of the individual female. The data reviewed here suggest that these sex-related differences in NADPH metabolism are a major cause of the higher incidence of liver cancer in female flounder in polluted environments.

Measuring Ca2+-signalling at fertilization in the sea urchin Psammechinus miliaris: alterations of this Ca2+-signal by copper and 2,4,6-tribromophenol.

During fertilization, eggs undergo a temporary rise in the intracellular concentration of free Ca(2+) ions. Using the membrane permeable acetoxymethylester of the fluorescent calcium indicator dye Fura-2, Fura-2 AM, the Ca(2+)-signal at fertilization was not detectable in eggs of the sea urchin Psammechinus miliaris. However, after treatment of the eggs with Fura-2 AM in combination with MK571, an inhibitor for multidrug resistance associated proteins, clear Ca(2+)-signals at fertilization could be measured without microinjection of the dye. We used this methodology to detect possible alterations of Ca(2+)-signalling at fertilization by exposure of eggs to environmental pollutants. For this purpose, the heavy metal copper, the bromophenol 2,4,6-tribromophenol, the organic compound bisphenol A and the polycyclic aromatic hydrocarbon phenanthrene were tested for their potential to inhibit fertilization success of P. miliaris. Copper and 2,4,6-tribromophenol showed a dose-dependent effect on fertilization rates of P. miliaris and significantly inhibited fertilization at 6.3 microM Cu(2+) and 1 microM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 microM while phenanthrene had no effect up to 56 microM. 6.3 microM copper and 100 microM 2,4,6-tribromophenol significantly increased the Ca(2+)-signal at fertilization. This alteration may contribute to the reduced fertilization rates of P. miliaris after exposure to copper and 2,4,6-tribromophenol.

In vivo inhibition of cysteine proteinases delays the onset of growth of human pancreatic cancer explants

An animal model was used to study the effects of oral treatment with a small molecular selective inhibitor of cysteine proteinases, Z-Phe-Arg-fluoromethylketone (Z-Phe-Arg-FMK) on primary tumour development. Poorly differentiated rapidly growing and moderately differentiated slowly growing human pancreatic tumours were implanted in the neck of nude mice that were orally treated or not with the inhibitor. Growth rates of the tumours were determined during 38 days after implantation. The poorly differentiated tumours were not affected by treatment with the inhibitor. Development of the moderately differentiated tumours was inhibited significantly by Z-Phe-Arg-FMK treatment. Moreover, the amount of stroma was increased and the volume of cancer cells was reduced in the moderately differentiated tumours that had grown in the treated animals. Reduction in size of the tumours was not achieved by reduction in growth rate but in a delay of the onset of growth. It is concluded that cysteine proteinases play a transient role at the start of tumour development only when cancer cells are surrounded by stroma as was the case in the moderately differentiated but not in the poorly differentiated pancreatic tumours. However, this role of cysteine proteinases can easily be taken over by other proteinases.

Immuno-localisations (GSSP) of subcellular accumulation sites of phenanthrene, aroclor 1254 and lead (Pb) in relation to cytopathologies in the gills and digestive gland of the mussel Mytilus edulis

Cell and tissue pathology of both, gill and digestive tissue, has been the subject of many studies for the elucidation of contaminant-induced biological effects. In the present study, cellular pathological alterations were linked to subcellular sites of chemical accumulation in gills and digestive gland tissues. For this purpose, mussels (Mytilus edulis) were exposed to the organic contaminants aroclor 1254 (PCB) (20 microg/L), phenanthrene (PAH) (150 microg/L) or the metal lead (Pb) (2.5mg/L). The localization of chemicals at the subcellular level was analysed by an antibody-based detection system (GSSP) by the use of commercially available antibodies specifically directed against the chemicals. Pathological changes were analysed in parallel in identical samples by transmission electron microscopy. After exposure to the different contaminants, cell organelles such as mitochondria, the endo-lysosomal system as well as endoplasmic reticulum showed clear evidence of chemically-induced alterations. Large numbers of crystalloid inclusions were found in mitochondria and in autophagic lysosomes as well as multi-lamellated whorls after PAH and aroclor exposure. Immunocytochemical detection of the chemicals showed their accumulation inside of various cell organelles such as lysosomes, mitochondria, and nuclei. Additionally, chemicals were localized in association to membranes, cilia and microvilli of gill and digestive gland cells. Furthermore, the chitinous rod and mucus secretions of gill epithelial cells were positively labelled for contaminants indicating their role in protection. Localization of contaminants by immuno-detection in combination with pathological diagnosis gives insights into the cellular targets of chemical attack.

Electron‐microscopic localization of lipophilic chemicals by an antibody‐based detection system using the blue mussel Mytilus edulis as a model system

Identification of lipophilic chemicals requires the development of new techniques to detect these compounds at the cellular and subcellular levels. To address this issue, we have developed a combinational protocol using antibodies directed against chemicals together with a multiple signal amplification system (catalyzed signal amplification/gold-substituted silver-intensified peroxidase) to detect the lipophilic compounds at their subcellular sites of accumulation by transmission electron microscopy. As a model organism, we exposed the blue mussel Mytilus edulis to the polycyclic aromatic hydrocarbon phenanthrene and the polychlorinated biphenyl mixture Aroclor 1254 for up to 10 d. The aim of these exposure experiments was to analyze the cellular targets and sites of accumulation in the digestive system by transmission electron microscopy. The endo-lysosomal system of digestive cells and mitochondria of epithelial cells appeared to be the preferred accumulation sites for the lipophilic compounds studied. The antibody-based approach to detect lipophilic chemicals provides an important insight into the pathways of uptake and accumulation as well as mechanisms of toxicity.

The liver of wrasse - morphology and function as a mirror of point source chemical impact.

Corkwing wrasse (Symphodus melops L.), a protogynous, non-migratory lipfish species, living close to rocky shores was chosen as an indicator species for the monitoring of biological effects of contaminants. Fish were caught by local fisherman at the Norwegian west coast in fjord sites within the framework of the EU BEEP project. The sites represented different point source impacts of (I) copper (a former copper mine), (II) polycyclic aromatic hydrocarbons (PAHs, aluminium smelter discharge), (III) formaldehyde plus PAHs (kelp-factory and influence of the aluminium smelter). Livers of wrasse were studied for histopathological alterations and compared to healthy livers of fish from a reference location. Besides liver morphology, different functional and metabolic parameters were measured to link pathological alterations to functional disorders. The integrity of the lysosomal compartment was tested by the assessment of lysosomal membrane stability (lys), and the accumulation of neutral lipids and lipofuscin. Activity and intracellular localisation of the NADPH-producing enzymes in the liver were assessed histochemically and measured by computer-assisted image analysis. Histopathological alterations were most severe at the site impacted by formaldehyde and PAHs. These findings were associated with highest tumor prevalence, lowest membrane stabilities in hepatocytes and highest accumulation rates of lipofuscin in the liver. The activities of the NADPH-producing enzymes phosphogluconate dehydrogenase (PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) were significantly lower compared to unimpacted reference fish. Histopathological alterations showed clear differences dependent on the input source. Potential links between specific contaminant impact and functional and morphological disorders are discussed.

Cellular localization of lead using an antibody‐based detection system and enzyme activity changes in the gills and digestive gland of the blue mussel Mytilus edulis

Marine organisms are continuously exposed to heavy metals in their environment. Bivalve mollusks such as the blue mussel Mytilus edulis accumulate high levels of heavy metals effecting cellular homeostasis and functions. Lead (Pb) exposure (2.5 mg/L of lead (II) nitrate for 10 d) and depuration (10 d in clean seawater) experiments were conducted to study its intracellular fate in the gills and digestive gland of M. edulis. For this purpose, an antibody-based detection method for ultrastructural localization and a subcellular fractionation approach for chemical analysis of Pb were used. In addition, effects of Pb on enzyme activities involved in oxyradical scavenging, such as the conjugative enzyme glutathione-S-transferase and the antioxidative enzyme catalase, were determined. The ultrastructural studies showed that Pb was mainly detected in lysosomes of gill epithelial cells and digestive cells. Lead was also detected in cell nuclei and granular hemocytes. Higher metal concentrations were measured by chemical analysis in subcellular fractions of the gills compared to those of the digestive gland. Increased activities of glutathione-S-transferase were found in gills after exposure and remained elevated during the depuration period, whereas glutathione-S-transferase activity remained unaffected in the digestive gland. Catalase activities showed no changes after Pb exposure, neither in the gills nor in the digestive gland. We conclude that gill cells are major sites of uptake and accumulation for dissolved Pb and are involved in sequestration and detoxification of this metal in M. edulis.