An extended study of seroprevalence of anti-Anisakis simplex IgE antibodies in Norwegian blood donors.

During the last decade, cases of the fish parasite Anisakis simplex infection and allergy in human have increased in countries with high fish consumption. Our aim was to perform an extended seroprevalence study of anti-IgE antibodies against this parasite in Norway, one of the high fish-consuming countries. At the Department of Immunology and Transfusion Medicine and the Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway, two main groups of anonymized serum samples were collected; the first (n = 993) from recently recruited blood donors (designated 'BDO') and the second (n = 414) from patient with total IgE levels ≥1000 kU/l (designated 'IGE+'). The sera were analysed by the ImmunoCAP(®) method for total IgE and IgE antibodies against A. simplex, house dust mite (HDM), shrimp, cod, crab, brine shrimp and shrimp tropomyosin. The A. simplex positive sera were further tested by an enzyme-linked immunosorbent assay (ELISA) method, which uses 2 recombinant (r) major allergens, rAni s 1 and rAni s 7 as target antigens. SDS-PAGE and Western immunoblotting analyses were also performed. Whereas the prevalences by ImmunoCAP(®) were 0.4% and 16.2% in the BDO and IGE+ groups, respectively, analyses with recombinant allergens showed only 0.0% and 0.2%. Cross-reactivity and immunoblotting analyses suggested that most of the ImmunoCAP(®) positive sera were probably false-positive due to cross-sensitization to shrimp and HDM. However, positivity due to other A. simplex antigens should also be considered. Compared with other high fish-consuming countries, we observed a very low seroprevalence of anti-Anisakis IgE antibodies in a Norwegian population.

Food allergens in mattress dust in Norwegian homes - a potentially important source of allergen exposure.

BACKGROUND: Sensitization to food allergens and food allergic reactions are mostly caused by ingesting the allergen, but can also occur from exposure via the respiratory tract or the skin. Little is known about exposure to food allergens in the home environment. OBJECTIVE: The objective of this study was firstly to describe the frequency of detection of allergens from fish, egg, milk, and peanut in mattress dust collected from homes of 13-year-old adolescents and secondly to identify home characteristics associated with the presence of food allergen contamination in dust. METHODS: Food allergens were measured by dot blot analysis in mattress dust from 143 homes in Oslo, Norway. We analysed associations between home characteristics (collected by parental questionnaires and study technicians) and food allergens by multivariate regression models. RESULTS: Fish allergen was detected in 46%, peanut in 41%, milk in 39%, and egg allergen in 22% of the mattress dust samples; only three samples contained none of these allergens. All four food allergens were more frequently detected in mattresses in small dwellings (< 100 m(2)) than larger dwellings (≥ 130 m(2)); 63-71% of the small dwellings (n = 24) had milk, peanut, and fish allergens in the samples compared with 33-44% of the larger dwellings (n = 95). Milk, peanut, and egg allergens were more frequently detected in homes with bedroom and kitchen on the same floor as compared with different floors, with odds ratios of 2.5 (95% confidence interval (CI): 1.1, 5.6) for milk, 2.4 (95% CI: 1.0, 6.1) for peanut, and 3.1 (95% CI: 1.3, 7.5) for egg allergens. CONCLUSIONS AND CLINICAL RELEVANCE: Food allergens occurred frequently in beds in Norwegian homes, with dwelling size and proximity of kitchen and bedroom as the most important determinants. Due to the amount of time children spent in the bedroom, mattress dust may be an important source of exposure to food allergens.

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta).

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole [(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole] and fenpropimorph [(+/-)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc] were administrated in the water separately and together in a static system (80 microg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole[2-(thiazol-4'-yl)benzimidazole] in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.

Effects of atrazine, endosulfan and butylated hydroxyanisole on glutathione-S-transferases in Orthosia gothica.

Atrazine (1,000 ppm), endosulfan (1 ppm) or butylated hydroxyanisole (BHA) (1,000 ppm) added to a semi-synthetic diet of Orthosia gothica for 2 days in the last instar did not change the soft tissue cytosolic glutathione-S-transferase (GST) activities towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and cumene hydroperoxide (CU). However, all three pesticides changed the GST subunit composition compared with the control as observed by reverse phase high performance liquid chromatography of the isozymes purified by glutathione-Sepharose affinity chromatography. The changes seem to have occurred mainly in the GST class 2 subunit. There is no obvious explanation for the changes, which may be a result of interactions between xenobiotic and GST in the cytosol as well as changes in the level of regulation of synthesis. However, the observation added to our knowledge of the processes involved when pesticides are degraded by GSTs in vivo.

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.

Time- and dose-dependent biom arker responses in flounder (Platichthys flesus L.) exposed to benzo[a]pyrene, 2,3,3',4,4', 5-hexachlorobiphenyl (PCB-156) and cadmium.

Responses in flounder (Platichthys flesus) towards benzo [a]pyrene (BaP), 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156), and cadmium (Cd) were investigated in time-course and dose-response studies of selected biomarkers. Measurements of biliary fluorescent BaP metabolites and hepatic concentrations of PCB-156 and cadmium showed that the injected toxicants were rapidly m obilized from the muscle to the liver, but a depot effect was indicated in the highest dose groups of BaP and PCB-156 (12 mg kg(-1) bodyweight). Clearest biomarker responses were found in the induction of hepatic cytochrome P450 1A (CYP1A) enzymes as a response towards BaP and PCB-156 exposure. Maximum induction of CYP1A dependent 7-ethoxyresorufin O-deethylase (EROD) activity was observed after 2 and 8 days in BaP and PCB-156-treated flounder, respectively. Positive dose-effect relationships were observed towards both compounds, but the CYP1A induction was more persistent with PCB exposure than with BaP exposure. In Cd-exposed fish, the hepatic level of metallothionein responded more slowly with highest levels observed after 16 days in the time-study. In the combined BaP + Cd treatment, the CYP1A induction was only slightly suppressed. Aspartate aminotransferase in serum appeared to be responsive towards BaP, but also towards the acetone vehicle in controls in the first part of the exposure period. Hematocrit as well as hepatic activities of aldrin epoxidase, glutathione S-transferase, and UDP-glucuronyl transferase were not responsive to any treatm ent in the present study. In general, the results demonstrate that selected biom arkers in flounder are responsive to PAH, PCB, and heavy metal pollutant exposure, indicating the applicability of this species in future environmental pollution monitoring programmes.

The separation and identification of glutathione S-transferase subunits from Orthosia gothica.

Four subunits of the cytosolic glutathione S-transferase (GST) in Orthosia gothica fed on willow leaves and a semisynthetic bean diet were purified as separate peaks (subunits 1-4) by a two-step gradient elution from a reverse-phase HPLC column after an initial purification by glutathione-Sepharose 1-chloro-2,4-dinitro-benzene (CDNB). Subunit 1 with a molecular weight of 26.0 kDa reconstituted into a GST homodimer with an isoelectric point of 4.8 and the N-terminal amino acid sequence (27 steps) indicated a relationship to the class theta GST of Musca domestica in the first 10 steps (50% homology), but also to the GST class pi of Caenohrabditis elegans (50% between steps 10 and 20). The three subunits 2-4 all had a molecular weight of 23.5 kDa and the isoelectric points of the reconstituted homodimers were > 9.0. The N-terminal amino acid sequence was determined (24 steps) and was identical for the three subunits. A high identity of sequence to the GST in C. elegans (70% between steps 1 and 17), and a low homology (25%) to the O. gothica subunit 1 was observed. Thus, we suggest the O. gothica subunit 1 belong to a different class (O. gothica GST class 1) of GST than subunits 2-4 (O. gothica GST class 2). When the larvae hatched and fed on a semisynthetic bean diet, subunits 3 and 4 were not present in the HPLC eluate, and the subunit 2/subunit 1 ratio increased compared to the corresponding ratio in the larvae which hatched and fed on willow leaves until the third instar.

Strain- and sex-specific differences in the glutathione S-transferase class pi in the mouse examined by gradient elution of the glutathione-affinity matrix and reverse-phase high performance liquid chromatography.

A gradient elution with glutathione (GSH) from a GSH-Sepharose 6B affinity column separated the hepatic mouse glutathione S-transferases (GST) to the alpha-, mu- and pi-classes. The GST-dependent conjugation of atrazine and glutathione was catalyzed by a pi-class GST. The pi- and mu-classes were both identified by their respective specific substrates, and after reverse-phase HPLC, by N-terminal analysis of 19-35 of the amino acids. The alpha-class GST was associated with a high selenium-independent GSH peroxidase activity and the purified protein had a N-blocked terminal. Strain related differences in the pi-class GST of the CD-1, C57BL/6, DBA/2 and Swiss-Webster males were observed by PhastGel electrophoresis of the GSH affinity chromatograph separated fractions, reverse phase HPLC and by N-terminal amino acid sequence analysis.

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.

A comparative study of effects of atrazine on xenobiotic metabolizing enzymes in fish and insect, and of the in vitro 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)

Response of xenobiotic metabolizing enzymes of rainbow trout (Oncorhynchus mykiss) to the mono-ortho substituted polychlorinated PCB congener 2,3',4,4',5-pentachlorobiphenyl, PCB-118, detected by enzyme activities and immunochemical methods.

The mono-ortho-substituted polychlorinated PCB congener 2,3',4,4',5'-pentachlorobiphenyl (PCB-118) was administered i.p. (30 mg/kg body weight) to gonadally immature rainbow trout (Oncorhynchus mykiss), of both sexes. In liver microsomes prepared from fish killed 4 days after administration, the cytochrome P450-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 P450 isozyme was determined with Western blotting and an enzyme-linked immunosorbent assay (ELISA) using rabbit anticod P450IA1 IgG. The monooxygenase parameters EROD and AHH were significantly induced to 558 and 268%, respectively, of the corresponding control values, while NCCR and AE activities were not affected. The antibodies to cod P450IA1 recognized a single protein band (Mr = 58,000 D) in the rainbow trout liver microsomes. The ELISA absorbance of this protein in the PCB-118 treated fish was 401% of the corresponding value in the controls. These results demonstrate that PCB-118 is an effective inducer of the subfamily cytochrome P450IA1 in rainbow trout liver microsomes.

Effects of polychlorinated biphenyls and environmental temperature on in vitro formation of benzo[a]pyrene metabolites by liver of trout (Salmo gairdneri).

Rainbow trout (Salmo gairdneri) held at 7 degrees and 16 degrees were given Aroclor 1254 (PCB) (10 mg/kg body wt) via intraperitoneal injections. The binding of [3H]benzo[a]pyrene (BaP) to deproteinized salmon sperm DNA was assayed (pmoles BaP equivalents per mg DNA per mg protein) using the post-mitochondrial supernatant (S 10) fractions from livers of fish at 24-168 hr after the PCB exposure. Liver enzymes from the untreated fish acclimated at 7 degrees yielded an average binding value (0.37 +/- 0.17) which was significantly greater (P less than 0.05) than the value (0.07 +/- 0.03) for untreated fish at 16 degrees. Liver supernatant fractions from PCB-induced fish acclimated at 16 degrees and sampled at 24-120 hr showed a substantial increase (P less than 0.05) in the binding (average value 2.4 +/- 1.8) compared to the value obtained with untreated fish at 16 degrees. At 24, 48 and 120 hr after the PCB treatment of fish held at 7 degrees, there was no significant increase in the binding value or extent of metabolism of BaP compared to that obtained with the untreated fish at 7 degrees. However, at 168 hr, three of four fish at 7 degrees responded to the PCB treatment with significantly (P less than 0.05) increased binding values (3.3 +/- 1.6). Chromatographic analyses of the ethyl acetate-soluble metabolites revealed that 3-hydroxy BaP and 7,8- and 9,10-dihydrodiols were the major metabolites; K-region metabolites were formed in trace amounts in untreated and PCB-treated fish at both temperatures. No marked qualitative differences were observed in metabolite profiles after the PCB treatment; however, overall metabolism of BaP and production of reactive metabolites by liver enzymes were considerably (P less than 0.05) enhanced in the PCB-induced fish at both 7 degrees and 16 degrees.

Qualitative and quantitative analyses of polychlorinated biphenyls by gas-liquid chromatography.

A model is presented of the relationship between the relative responses of flame-ionization and electron-capture detectors and the structure of polychlorinated biphenyls (PCBs). The model permits the calculation of detector responses for all PCBs and opens the possibility of detailed structural analyses.

Naturally occurring vitamin D3 in fish products analysed by HPLC, using vitamin D2 as an international standard.

A chemical method for the analysis of naturally occurring vitamin D is proposed. The unsaponifiable matter of oils and tissues is prepared, cholesterol is partly removed by double precipitation at low temperature in methanol. The vitamin D fraction is collected on an adsorption column by high performance liquid chromatography. The fraction is further purified and the vitamins D2 and D3 are separated on a partition column (reverse phase) by HPLC. Recovery was 89 to 93%, standard deviation 3%. The only vitamin D analogue found in fish oils, livers and fillets, was cholecalciferol (D3). Hence, ergocalciferol (D2) could be used as an internal standard. The provitamins ergosterol and 7-dehydrocholesterol, as well as the previtamins, were separated from the vitamin D-fraction on the adsorption column. Results in the range 0.050 to 134 microgram D3 per gram (2 to 5360 I.U. per gram) are given. One cod liver oil was analysed in a rat bioassay, giving supporting results.