Constanze Pietsch

Endocrine disruption in aquatic vertebrates.

Environmental compounds can interfere with endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disrupters (ED), are surface waters. Thus, aquatic vertebrates, such as fish and amphibians, are most endangered. ED can adversely affect reproductive biology and the thyroid system. ED act by (anti)estrogenic and (anti)androgenic modes of action, resulting in abnormal sexual differentiation and impaired reproduction. These effects are mainly driven by direct interferences of ED with sex steroid receptors rather than indirectly by impacting synthesis and bioavailability of sex steroids, which in turn might affect the hypothalamic–pituitary–gonadal axis. Recent findings reveal that, in addition to the human‐produced waste of ED, natural sources, such as parasites and decomposition of leaves, also might act as ED, markedly affecting sexual differentiation and reproduction in fish and amphibians. Although the thyroid system has essential functions in both fish and amphibians, amphibian metamorphosis has been introduced as the most sensitive model to detect thyroidal ED; no suitable fish model exists. Whereas ED may act primarily on only one specific endocrine target, all endocrine systems will eventually be deregulated as they are intimately connected to each other. The recent ecotoxicological issue of pharmaceutically active compounds (PhACs) present in the aquatic environment indicates a high potential for further endocrine modes of action on aquatic vertebrates by ED derived from PhACs, such as glucocorticoids, progestins, and ß‐agonists.

Occurrence of deoxynivalenol and zearalenone in commercial fish feed: an initial study.

The control of mycotoxins is a global challenge not only in human consumption but also in nutrition of farm animals including aquatic species. Fusarium toxins, such as deoxynivalenol (DON) and zearalenone (ZEN), are common contaminants of animal feed but no study reported the occurrence of both mycotoxins in fish feed so far. Here, we report for the first time the occurrence of DON and ZEN in samples of commercial fish feed designed for nutrition of cyprinids collected from central Europe. A maximal DON concentration of 825 μg kg−1 feed was found in one feed whereas average values of 289 μg kg−1 feed were noted. ZEN was the more prevalent mycotoxin but the concentrations were lower showing an average level of 67.9 μg kg−1 feed.

Effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings

A 56-day feeding trial was conducted to access the effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings. Seven feeds formulated to contain 36% protein and 20 kJ g(-1) gross energy (dry matter basis), were prepared by replacing fish meal with magmeal. Fifteen fingerlings (initial average weight 2.0 +/- 0.1 g) stocked per experimental tank were fed in triplicates at 5% body weight in two portions per day (a level previously established). Growth and food conversion ratio were adequate and comparable without any significant differences (p < 0.5) between feeding groups. Mean values for haematocrit and plasma glucose were not significantly different (p < 0.05) among the feeding groups. Fish group fed control diet (containing highest inclusion level of fish meal and without magmeal) gave the lowest haemoglobin concentration (5.96 +/- 0.22 g dl(-1)). This value was significantly different from other feeding groups. Stressful conditions in fish and in mammals are associated with decreased growth, haematocrit (packed cell volume) and haemoglobin values, increased whole blood glucose (hyperglycaemia) and plasma cortisol concentrations. No such physiological changes were observed in this study. Results suggest that feeding O. niloticus fingerling with magmeal diets did not cause any form of physiological stress. Magmeal can be used as a good alternative protein source in tilapia diets.

Frequent biphasic cellular responses of permanent fish cell cultures to deoxynivalenol (DON)

Contamination of animal feed with mycotoxins is a major problem for fish feed mainly due to usage of contaminated ingredients for production and inappropriate storage of feed. The use of cereals for fish food production further increases the risk of a potential contamination. Potential contaminants include the mycotoxin deoxynivalenol (DON) which is synthesized by globally distributed fungi of the genus Fusarium. The toxicity of DON is well recognized in mammals. In this study, we confirm cytotoxic effects of DON in established permanent fish cell lines. We demonstrate that DON is capable of influencing the metabolic activity and cell viability in fish cells as determined by different assays to indicate possible cellular targets of this toxin. Evaluation of cell viability by measurement of membrane integrity, mitochondrial activity and lysosomal function after 24 h of exposure of fish cell lines to DON at a concentration range of 0-3000 ng ml(-1) shows a biphasic effect on cells although differences in sensitivity occur. The cell lines derived from rainbow trout are particularly sensitive to DON. The focus of this study lies, furthermore, on the effects of DON at different concentrations on production of reactive oxygen species (ROS) in the different fish cell lines. The results show that DON mainly reduces ROS production in all cell lines that were used. Thus, our comparative investigations reveal that the fish cell lines show distinct species-related endpoint sensitivities that also depend on the type of tissue from which the cells were derived and the severity of exposure.

Organ Damage and Hepatic Lipid Accumulation in Carp (Cyprinus carpio L.) after Feed-Borne Exposure to the Mycotoxin, Deoxynivalenol (DON)

Deoxynivalenol (DON) frequently contaminates animal feed, including fish feed used in aquaculture. This study intends to further investigate the effects of DON on carp (Cyprinus carpio L.) at concentrations representative for commercial fish feeds. Experimental feeding with 352, 619 or 953 μg DON kg−1 feed resulted in unaltered growth performance of fish during six weeks of experimentation, but increased lipid peroxidation was observed in liver, head kidney and spleen after feeding of fish with the highest DON concentration. These effects of DON were mostly reversible by two weeks of feeding the uncontaminated control diet. Histopathological scoring revealed increased liver damage in DON-treated fish, which persisted even after the recovery phase. At the highest DON concentration, significantly more fat, and consequently, increased energy content, was found in whole fish body homogenates. This suggests that DON affects nutrient metabolism in carp. Changes of lactate dehydrogenase (LDH) activity in kidneys and muscle and high lactate levels in serum indicate an effect of DON on anaerobic metabolism. Serum albumin was reduced by feeding the medium and a high dosage of DON, probably due to the ribotoxic action of DON. Thus, the present study provides evidence of the effects of DON on liver function and metabolism.

In vivo effects of deoxynivalenol (DON) on innate immune responses of carp (Cyprinus carpio L.).

Deoxynivalenol (DON) is one of the most important members of Fusarium toxins since it often can be found in relevant concentrations in animal feeds. The effects of this group of toxins on fish are mostly unknown. The present study shows results from a feeding trial with carp (Cyprinus carpio L.) using three different concentrations of DON (352μgkg(-1), 619μgkg(-1), and 953μgkg(-1) final feed, respectively) which are comparable to levels found in commercial fish feeds. Effects on growth and mass of fish were not observed during this 6weeks lasting experiment. Only marginal DON concentrations were found in muscle and plasma samples. Blood parameters were not influenced although smaller erythrocytes occurred in fish treated with 352μgkg(-1) DON. Analysis of antioxidative enzymes in erythrocytes showed increased superoxid dismutase and catalase activities in fish fed the low-dose feed. Immunosuppressive effects of DON were confirmed whereby cytotoxic effects on immune cells only partly explained the impairment of innate immune responses. Exact polarization of the immune system into pro-inflammatory or anti-inflammatory responses due to DON exposure should be clarified in further experiments, especially since the current results raise concern about impaired immune function in fish raised in aquaculture.

Effects of Dietary Exposure to Zearalenone (ZEN) on Carp (Cyprinus carpio L.)

The mycotoxin zearalenone (ZEN) is frequently contaminating animal feeds including feed used in aquaculture. In the present study, the effects of dietary exposure to ZEN on carp (Cyprinus carpio L.) were investigated. ZEN at three different concentrations (low dose: 332 µg kg−1, medium dose: 621 µg kg−1 and high dose: 797 µg kg−1 final feed, respectively) was administered to juvenile carp for four weeks. Additional groups received the mycotoxin for the same time period but were fed with the uncontaminated diet for two more weeks to examine the reversibility of the ZEN effects. No effects on growth were observed during the feeding trial, but effects on haematological parameters occurred. In addition, an influence on white blood cell counts was noted whereby granulocytes and monocytes were affected in fish treated with the medium and high dose ZEN diet. In muscle samples, marginal ZEN and α-zearalenol (α-ZEL) concentrations were detected. Furthermore, the genotoxic potential of ZEN was confirmed by analysing formation of micronuclei in erythrocytes. In contrast to previous reports on other fish species, estrogenic effects measured as vitellogenin concentrations in serum samples were not increased by dietary exposure to ZEN. This is probably due to the fact that ZEN is rapidly metabolized in carp.

Natural Mineral Particles Are Cytotoxic to Rainbow Trout Gill Epithelial Cells In Vitro

Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L−1) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H2DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8–1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6–1.8-fold-changes at the 250 mg L−1 concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3–1.6-fold increases at the 250 mg L−1 concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with clay particles being more cytotoxic, and (iii.) some clays might induce effects comparable to engineered nanoparticles.

Production of nitric oxide by carp (Cyprinus carpio L.) kidney leukocytes is regulated by cyclic 3',5'-adenosine monophosphate

The inducible nitric oxide synthase (iNOS) plays a central role in the inflammatory reactions that follow infection or tissue damage. Induction of nitric oxide (NO) synthesis by bacterial lipopolysaccharide (LPS) depends on activation of G protein-coupled receptors in mammals. Thus, it was our intention to evaluate whether similar mechanisms are involved in iNOS activation in fish leukocytes. Therefore, the participation of membrane-bound receptors which activate effectors via G proteins has been confirmed using the G protein inhibitor suramin. Furthermore, the NO produced by iNOS performs both beneficial and detrimental actions. It is thus conceivable that regulatory mechanisms exist which control the timing and intensity of NO production by iNOS in order to outweigh protective effects against detrimental ones. The second messenger cAMP produced by adenylyl cyclases (ACs) plays a key role in the regulation of many cellular functions. Since cAMP signaling inhibits numerous immunological reactions, studies have been carried out to determine whether cAMP-dependent pathways could inhibit NO production by carp leukocytes as well. To measure cellular responses such as NO production by carp leukocytes derived from head and trunk kidneys treatments were performed with the cAMP elevating agents forskolin and dibutyryl-cAMP (db-cAMP) prior to stimulation with Aeromonas hydrophila. Pharmacological studies in stimulated kidney leukocytes showed that increased intracellular cAMP levels lead to reduced NO formation. This reduction of NO production was not due to decreased cell numbers, since a tetrazolium dye-based assay revealed no reduction of cell viability by cyclic nucleotide elevating agents. Thus, our data provide evidence that the AC/cAMP signaling pathway is well established in carp leukocytes. Cyclic AMP leads to type II immune response. We provide evidence that the predominant AC in fish leukocytes is a particulate enzyme due to its sensitivity to forskolin. Treatment of leukocytes with agents increasing intracellular cAMP gave clear evidence for participation of this cyclic nucleotide in immune signaling.

Cytotoxic effects of pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCHQ) on liver cells are modulated by antioxidants

The worldwide distribution and high bioaccumulation potential of pentachlorophenol (PCP) in aquatic organisms imply a high toxicological impact in aquatic systems. Firstly, our investigations show that, similar to mammalian cell lines, PCP can be metabolized to tetrachlorohydroquinone (TCHQ) in the permanent cell line derived from rainbow trout liver cells (RTL-W1). Moreover, we demonstrate that PCP as well as its metabolite TCHQ is capable of influencing the viability of these cells. Three cell viability assays were performed to assess possible cellular targets of these substances. Thus, the cytotoxicity of the PCP-derivative TCHQ was shown for the first time in a fish cell line. Further investigations revealed the involvement of ROS in the cytotoxicity of PCP and its metabolite TCHQ. The observation of oxidative stress provides a plausible explanation for the increased cytotoxicity at higher concentrations especially for PCP and implies possible mechanisms underlying these observations. In addition, antioxidants such as ascorbic acid and quercetin modulate the detrimental effects of PCP and TCHQ whereby both compounds exacerbate the cytotoxic effects of high PCP and TCHQ concentrations.