Eliann Egaas

Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) exposed by caging to polluted sediments in Sørfjorden, Norway

Flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) were subjected to caging at polluted sediments in a Norwegian fjord (Sorfjorden) for a period of 3 months. Three caging sites were located close to metal smelters, whereas a fourth site was located 30 km away as a reference. In sediment samples, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals were elevated at the innermost sites (1, 2 and 3) compared with the reference location (site 4). In fish, the biliary levels of fluorescent aromatic compounds (FACs) were elevated 5–20 fold in both species at the polluted sites. A two-fold difference in heavy metal levels was observed in cod (site 2 vs. 4), whereas no differences were seen in flounder. Pesticides bioaccumulated in a diffuse manner at all sites. In flounder at the innermost sites, plasma aspartate aminotransferase (AST) and hepatic cytochrome P450 1A (CYP1A) dependent 7-ethoxyresorufin O-deethylase (EROD) activity were elevated 4–5 and 5–10 fold, respectively, compared with the reference site. Both of these biomarkers were significantly correlated with FACs levels. For other biomarkers, the site effect was more marginal. The biomarkers seemed in general more responsive in flounder than in cod. The present study demonstrates biomarker measurements in caged fish as a promising approach for evaluating accumulation and effects of pollutants in marine teleosts.

Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta)

The fungicide propiconazole (1-(2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl) -1H-1,2,4-triazole) induced the hepatic cytochrome P4501A (CYP1A) activity towards ethoxyresorufin-O-deethylase (EROD), the content of CYP1A protein as quantified by enzyme-linked immunosorbent assay (ELISA) and the glutathione S-transferase (GST) activity towards the three commonly used substrates CDNB(1-chloro-2,4-dinitrobenzene), cumene hydroperoxide (CU) and ethachrynic acid (EA) in brown trout (Salmo trutta) depending on dose and body weight. An exponential dose response relationship existed between propiconazole exposure and CYP1A activity. A 2. order polynomial regression of the propiconazole concentration (square root transformed) on the data for CDNB, EU and CU revealed a bell-shaped pattern of the GST induction. Reverse-phase HPLC of the GSH-affinity chromatography purified GST isozymes in trout exposed to respectively 8.3, 23, 93, 313 and 606 microg l(-1) propiconazole in the water indicated that the propiconazole treatment may lead to changes in the composition of the subunits compared to the controls. Thus, propiconazole exposure through the water changed the properties of the brown trout hepatic CYP1A and GST, and these changes may be used as a bioindicator on the molecular level of exposure and effect of propiconazole in controlled experiments. The use in monitoring of propiconazole exposure under natural field conditions is possible, however needs further investigation.

A comparative study of effects of atrazine on xenobiotic metabolizing enzymes in fish and insect, and of the invitro phase II atrazine metabolism in some fish, insects, mammals and one plant species

1. Atrazine (3 daily i.p. doses of 0.20 mg/kg or 10 ppb in the water for 14 days) did not change the xenobiotic metabolizing enzyme activities (XME) towards the substrates aldrin epoxidase (AE), NADPH-cytochrome c reductase (NCCR), 7-ethoxyresorufin O-deethylase (EROD), 1-chloro-2,4-dinitro-benzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) in trout liver (Oncorhynchus mykiss) compared to the controls. 2. Various treatment regimens of atrazine in a semisynthetic diet changed the XME activities towards AE, NCCR, CDNB and DCNB in the cabbage moth (Mamestra brassica L.) soft tissues and midgut compared to the controls. 3. A life-long cabbage diet induced the XME activity towards CDNB in the cabbage moth soft tissues and midgut, whereas no differences in the activities towards AE, NCCR and DCNB were observed compared to controls on a semi-synthetic diet. 4. The cabbage moth GSTs bound poorly to a glutathione (GSH)-linked epoxy-activated Sepharose 6-B; however, based on the CDNB activity recovered by a GSH elution, there were no differences in the molecular weights of the partly purified subunits (27, 26 and 25 kDa) or the pIs (5.4, 4.8, and 4.1) of the molecules in the soft tissues or midguts from respectively atrazine treated and control cabbage moth.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers in flounder (Platichthys flesus): an evaluation of their use in pollution monitoring

Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study.

A case of peanut cross-allergy to lupine flour in a hot dog bread

Background: In a case monitored by the Norwegian National Register for Severe Allergic Reactions to Food, a patient with peanut allergy experienced an allergic reaction after eating a particular brand of hot dog bread. The aim of this study was to identify the eliciting allergen. Methods: Extracts from the hot dog bread and reference material from peanut, lupine and lupine-fortified food products were analysed by immunochemical methods with patient serum and a new polyclonal anti-lupine antibody. Results: Evidence could be provided that the hot dog bread contained proteins from lupine but not from peanut. Conclusion: Crossed peanut-lupine allergy can have clinical significance. A peanut-allergic patient reacted against hidden lupine protein in a hot dog bread. Presented with our results, the producer confirmed the use of lupine flour and changed the ingredient list.

Changes in humoral responses to β-lactoglobulin in tolerant patients suggest a particular role for IgG4 in delayed, non-IgE-mediated cow's milk allergy

The major cows milk allergen β‐lactoglobulin (β‐LG) is relatively resistant to enzymatic degradation and may therefore be involved in non‐immunoglobulin (Ig)E‐mediated cows milk allergy (CMA) with delayed gastrointestinal symptoms. Serum levels of β‐LG‐specific IgG1, IgG4, IgE, and IgA were compared in clinically reactive and tolerized IgE‐mediated and non‐IgE‐mediated CMA with delayed gastrointestinal symptoms (n = 29) and controls (n = 10). Tolerance was associated with decreased β‐LG‐specific IgE, IgG1, and IgG4 levels in both patient groups. However, the significantly increased β‐LG‐specific IgG4 levels in clinically reactive non‐IgE‐mediated CMA patients, and its median 36‐fold reduction in tolerant patients, suggested a possible immunopathological role for IgG4 in delayed CMA. Similarly, the significantly increased β‐LG‐specific IgE levels in IgE‐mediated CMA patients were decreased 44‐fold in tolerant patients. The tolerant patients had apparently shifted the humoral immune response from a β‐LG‐specific IgE‐ and/or IgG4‐dominated immune response to an IgA‐dominated immune response as the IgA/IgE or IgA/IgG4 ratios increased 90‐ and 15‐fold in tolerant IgE‐mediated‐, and non‐IgE‐mediated CMA patients, respectively. Thus, the marked difference in β‐LG‐specific Ig ratios suggested a tolerance‐induced inhibition of a Th2‐type of immune response with significantly increased IgA dominance in both CMA patient groups.

Interaction of benzo[a]pyrene, 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156) and cadmium on biomarker responses in flounder (Platichthys flesus L)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg-1), 2,3,3′,4,4′5 hexachlorobiphenyl (PCB-156, 2.5 mg kg-1), and cadmium (cadmium, 1 mg kg-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with...

A study of gender, strain and age differences in mouse liver glutathione-S-transferase

The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-1, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.

Memory T Cell Proliferation in Cow’s Milk Allergy after CD25+ Regulatory T Cell Removal Suggests a Role for Casein-Specific Cellular Immunity in IgE-Mediated but Not in Non-IgE-Mediated Cow’s Milk Allergy

Background: Previously reported increased lymphocyte proliferative responses in cow’s milk allergy (CMA) may have been influenced by the lipopolysaccharides (LPS) which contaminate most commercial cow’s milk protein (CMPs). Moreover, peripheral blood mononuclear cells (PBMC) contain both B cells, CD45RA+ naïve T cells, CD25+ regulatory T cells (Tregs) in addition to antigen-specific CD45RA– memory T cells. Methods: PBMC from clinically reactive and tolerised patients with IgE- and non-IgE-mediated CMA were depleted of CD45RA+ T cells and putative CD25+ Tregs. The proliferative index to LPS-depleted α-, β- and ĸ-casein and β-lactoglobulin was compared in the memory T-cell-enriched, Treg-depleted PBMC and in bulk PBMC. Results: Clinically reactive IgE-mediated CMA patients had increased responses to caseins only. Tolerised patients, particularly those with atopic dermatitis, had decreased responses to ĸ-casein which were restored after Treg depletion. Interleukin-4 and interferon-γ were generally not detected in the culture supernatants. No differences were seen between reactive and tolerant delayed non-IgE-mediated CMA patients. Conclusions: Proliferative responses to α-, β- and ĸ-caseins (but not β-lactoglobulin) were observed in clinically reactive IgE-mediated CMA patients only. A markedly decreased proliferative response to ĸ-casein in tolerised IgE-mediated CMA patients with atopic dermatitis, which was abrogated by Treg depletion, suggested a role for ĸ-casein in tolerance induction. Non-IgE-mediated CMA patients had no increased proliferative response to any milk proteins.

Response of xenobiotic metabolizing enzymes in rainbouw trout (Oncorhynchus mykiss) to endosulfan, detected by enzyme activities and immunochemical methods

Abstract A single i.v. injection (75 μg/kg body weight) of the insecticide endosulfan was administered to gonadally immature rainbow trout (Oncorhynchus mykiss). Experiments were done with both technical and analytical grade endosulfan. In liver microsomes prepared from fish killed 24 h after administration, the cytochrome P-450-dependent monooxygenase activities of 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), and aldrin epoxidase (AE) were measured. In addition, NADPH-cytochrome c reductase (NCCR) was analyzed, and the content of a specific cytochrome P−450 isozyme was determined with Western blotting and an enzyme-linked immunosorbent assay (ELISA) using rabbit anticod P−4501A1 IgG. Technical grade endosulfan significantly induced EROD and AE to 160 and 144%, respectively, of the corresponding control values, while analytical grade endosulfan significantly increased only AE activity to 147% and tended to induce EROD to 162%. AHH and NCCR were not affected. The antibodies to cod P−4501A1 recognized a single protein band (M1 = 58 Da) in the rainbow trout liver microsomes. The ELISA absorbances of this protein in the technical and analytical grade endosulfan treated fish were 151 and 138%, respectively, of the corresponding values in the controls. These results were supported by corresponding results from a 14 days study, where rainbow trout were exposed to technical grade endosulfan (8.3 μg/l water). In this study glutathione S-transferase (GST) activity towards 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also included, however, no effect on GST was observed.