Kristian Ingebrigtsen

Response of hepatic xenobiotic metabolizing enzymes in rainbow trout (Oncorhynchus mykiss) and cod (Gadus morhua) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD)

Abstract 2,3,7,8-Tetrachlorodibenzo- p -dioxin (2,3,7,8-TCDD) was administered intragastrically twice with 4 days interval, to juvenile cod and rainbow trout, total dose 8 μg/kg body weight. Fish were killed after 9 and 17 days and the effects on hepatic xenobiotic metabolizing enzymes were determined by examining aldrin epoxidase (AE), glutathione-S-transferase (GST) against 1-chloro-2,4-dinitrobenzene (CDNB) and the cytochrome P450-dependent ethoxyresorufin-O-deethylase (EROD) activities, and by immunoquantitating cytochrome P4501A1 using an indirect enzyme-linked immunosorbent assay (ELISA). AE and GST activities were not induced. However, 2,3,7,8-TCDD significantly induced EROD activities in rainbow trout and cod to 1450% and 415%, respectively, of the corresponding controls 9 days after the first treatment. The increase in EROD activity was supported by induction of the main catalyst P4501A1 as revealed by ELISA analyses. The distribution pattern of 14 C-labelled 2,3,7,8-TCDD was studied by whole-body autoradiography. The liver concentration of radiolabelled compound in cod exceeded that of the rainbow trout. However, the degree of hepatic enzyme induction did not correspond to the concentration of radiolabelled 2,3,7,8-TCDD in the liver. Despite of the substantially higher level of 2,3,7,8-TCDD in the liver of cod, the EROD induction was lower in this species compared to rainbow trout.

Cell‐specific CYP1A expression and benzo[a]pyrene adduct formation in gills of rainbow trout (Oncorhynchus mykiss) following CYP1A induction in the laboratory and in the field

The effect of cytochrome P4501A (CYP1A) induction on cell-specific benzo[a]pyrene (BaP) adduct formation was studied in rainbow trout (Oncorhynchus mykiss) gills. Fish preexposed to beta-naphthoflavone (betaNF) or caged in a polluted river were exposed to waterborne 3H-benzo[a]pyrene (3H-BaP). The 3H-benzo[a]pyrene adducts in the gill filaments were localized by autoradiography and CYP1A protein by immunohistochemistry. Ethoxyresorufin O-deethylase (EROD) activity was measured using a gill filament-based ex vivo assay. Branchial 3H-BaP binding and EROD activity were enhanced by exposure to betaNF or to the river water, and completely blocked by the CYP1A inhibitor ellipticine. The predominant sites of adduct formation were in epithelium of the secondary lamellae and in epithelium of the efferent edge of the gill filament. In betaNF-exposed fish, the strongest CYP1A immunoreactivity was observed in differentiating cells and in pillar cells. In fish caged in the polluted river, strong CYP1A immunoreactivity was found in most cells in the secondary lamellae, whereas the primary lamellae were almost devoid of immunoreactivity. Our results reveal a discrepancy between the localization of CYP1A protein and BaP adducts in the gill. Consequently, other factors, such as bioavailability of waterborne polycyclic aromatic hydrocarbons (PAHs) to the target cells, are important for the localization of PAH adducts in the gill.

Pharmacological studies of ricin in mice and humans.

SummaryA highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of ricin in serum is presented. Using this method it was found that IV-injected ricin disappeared from the plasma of mice and cancer patients according to first-order kinetics.DBA mice were found to be more sensitive to ricin than C3H and B6D2 mice. When mice of the different strains were given the same dose of ricin, the concentrations found in liver, spleen, and kidneys were highest in the most sensitive mice. Ricin disappeared most rapidly from serum of the mice with the highest sensitivity. The inverse correlation between the rate of disappearance of ricin from serum and the tissue concentrations reached may be due to the fact that ricin is rapidly and firmly bound to cell surface receptors.Whole-body autoradiography after IV injection of 125I-labeled ricin showed the highest amount of radioactivity in liver, spleen, and adrenal cortex. Considerable amounts of radioactivity were also present in bone marrow, showing that the lack of myelosuppressive activity of ricin previously found in mice and dogs cannot be accounted for by the failure of ricin to reach the bone marrow.Part of the ricin in the tissues was present in the form of free chains, the highest fraction being present in the liver.In this tissue both the free A-chains and those present in whole ricin were found to be modified. However, the modified A-chains had retained their full capacity to inhibit protein synthesis in vitro.In cancer patients, toxicity appeared at about the same initial serum levels as in the mice, supporting the view that mouse data have a good predictive value for man. At each dose level the individual variations were modest, a finding that is important for eventual clinical use of this potent drug.

Tissue distribution of 14C-astaxanthin in the Atlantic salmon (Salmo salar)

Abstract Atlantic salmon ( Salmo salar ) were fed a diet containing 14 C-labelled astaxanthin for five subsequent days. Ten days after the last feeding, two fish were sampled and submitted to whole-body autoradiography. A high degree of radioactivity was present in the dorsal cutis, the bile, the intestinal mucosa, the caudal kidney and the developing eggs. An intermediate degree of radiolabelling was found in the muscle and the cranial kidney, while only traces of radioactivity were recorded in the blood, the spleen and the gills. Contrary to the prevailing concensus, the level of astaxanthin and/or metabolites was higher in the myocommata than in the myotome. Furthermore, the substantial radiolabelling in the dorsal cutis strongly suggests that astaxanthin and/or its metabolites show affinity for melanin. Finally, the results give evidence for urinary and biliary excretion of astaxanthin-derived metabolites and for the likelihood of enterohepatic circulation.

Placental transfer of the estrogenic mycotoxin zearalenone in rats

In order to study the possible placental transfer of the Fusarium mycotoxin zearalenone (ZON), Sprague Dawley rats were treated with a single dose (0.74 mg/kg b.w.) of ZON i.v. on day 12 or day 18 of pregnancy, or intragastrically (i.g.) on day 18 of pregnancy. Samples of placenta, foetus, and maternal liver and spleen were collected for chemical analyses 0.3 h after treatment on day 12, and 0.3, 4, and 24 h after treatment on day 18. Three rats were used for each pregnancy day, administration route, and exposure time. The concentrations of ZON and its metabolites alpha- and beta-zearalenol (-ZOL) were determined quantitatively by high-performance liquid chromatography (HPLC) after incubation with beta-glucuronidase and purification on immunoaffinity columns. Tissue distribution was studied by means of whole body autoradiography at 4 and 24 h after treatment with tritiated ZON (750 microCi/kg b.w; 7.4 mg/kg b.w.) on day 18 of pregnancy. ZON and alpha-ZOL were transferred into the foetus on both gestational days. However, a delay in distribution into the foetus, relative to the maternal tissue, was observed. Beta-ZOL was below the detection limit in the foetus. No specific site of foetal accumulation of ZON or its metabolites was apparent. In the maternal tissues, the highest levels of ZON and of alpha- and beta-ZOL were found in the liver.

Distribution and elimination of [14C] hexachlorobenzene after single oral exposure in the rainbow trout (Salmo gairdneri).

Distribution and elimination of hexachlorobenzene (HCB) after administration to rainbow trout (Salmo gairdneri) of a single oral dose of 5 microCi [14C] HCB/100 g body weight were studied by whole-body autoradiography and liquid scintillation counting. To obtain some information on the physicochemical properties of the radiolabelled compounds, whole-body autoradiography was performed exposing parallel sagittal sections, treated at -20 degrees C, evaporated at 50 degrees C, and extracted separately with polar and nonpolar solvents. At d 1, radioactivity was distributed throughout the body. The highest concentration of radioactivity was found in adipose tissue. In the abdominal fat, the peak level of radioactivity was measured at d 30. No part of the radioactivity in the bile was evaporable. Radioactivity in the intestinal content, the skin, and the uveal tract was partly evaporable, while only traces of radioactivity remained in adipose tissue after evaporation. Radioactivity in the bile was extractable with water only. No radioactivity remained in any tissue after extraction with polar and nonpolar solvents. The rate of elimination was slow, and substantial amounts of radioactivity remained in the body 120 d after administration. In addition to bile excretion of nonevaporable, water-soluble radiolabeled compounds, a possible excretion over the intestinal mucosa was suggested.

Disposition and cellular binding of 3H-benzo(a)pyrene at subzero temperatures: studies in an aglomerular arctic teleost fish – the polar cod (Boreogadus saida)

Abstract Autoradiography at different levels of resolution was used to study the disposition of the polycyclic aromatic hydrocarbon benzo(a)pyrene (3H-BaP) in juvenile and sexually mature polar cod (Boreogadus saida). Exposure took place via the water or after intragastric administration at subzero temperatures. In water-exposed fish, high total tissue levels were found in the gills, olfactory organ, anterior kidney, liver, skin and intestinal wall. Only traces of radioactivity were present in the muscle, brain and gonads. No major differences in tissue levels or in general distribution pattern between males, females or juvenile fish were observed. The gills appeared to be the absorption site for exposure via water. After oral administration, tissue levels of 3H-BaP-derived radioactivity were negligible. Following both administration routes, levels of radioactivity were highest in the bile and intestinal contents while only traces were observed in the urine, indicating biliary excretion as the major excretory pathway in this aglomerular species. Tape-section autoradiography of fish exposed via water revealed tissue-bound residues of 3H-BaP in the olfactory organs, gills, kidney, liver, skin and intestinal mucosa. Light-microscopy autoradiography demonstrated that the bound residues in the olfactory organ, gills and anterior kidney were localized in epithelial cells, while those in liver and intestinal mucosa were evenly distributed. In conclusion, the present study shows that BaP is absorbed from the water via the gills at subzero temperatures, that tissue levels are considerably higher after water exposure than after dietary exposure, that biliary excretion is predominant and, finally, that site-specific tissue binding in the olfactory organs, gills and anterior kidney is confined to epithelial cells.

Distribution and elimination of [14C]-hexachlorobenzene after single oral exposure in cod (Gadus morhua) and flounder (Platichthys flesus).

Distribution and elimination of hexachlorobenzene (HCB) after administration to COD (Gadus morhua) of a single oral dose of 5 microCi [14C]HCB/100 g body weight were studied by whole-body autoradiography and liquid scintillation counting. To obtain some information on the physicochemical properties of the radiolabeled compounds, whole-body autoradiography was performed exposing parallel sagittal sections, treated at -20 degrees C, evaporated at 50 degrees C, and extracted separately with polar and nonpolar solvents. The highest concentration of radioactivity was found in the liver, the bile, and the central nervous system (CNS). Radioactivity in the liver and CNS, which was completely evaporable, was considered to represent the highly volatile HCB itself, and/or metabolites with high vapor pressure. No part of radioactivity in bile was evaporable, but it was completely extractable with water. Radioactivity in the intestinal content, the skin, and the uveal tract of the eye was partly evaporable. No radioactivity remained in any tissue after extraction with polar and nonpolar solvents. The rate of elimination was slow, and substantial amounts remained in the body 60 d after administration. In addition to bile excretion of nonevaporable, water-soluble radioactivity, a possible excretion through the intestinal mucosa was suggested. Whole-body autoradiography of female flounders (Platichthys flesus) revealed a high content of radioactivity in the developing eggs.

Comparison of dietary and waterborne exposure to benzo (a) pyrene: bioavailability, tissue disposition and CYP1A1 induction in rainbow trout ( Oncorhynchus mykiss)

Absorption and tissue distribution of benzo\ [a] pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.

Elimination of polychlorinated dibenzofurans and dibenzo-p-dioxins from blue mussel (Mytilus edulis) and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD)

Abstract Blue mussels ( Mytilus edulis ) were exposed to water supplied from a tank with sediment contaminated by polychlorinated dibenzofurans and dibenzo-p-dioxins (PCDF/PCDD). After an exposure period of 99 days the elimination patterns of PCDF/PCDD were followed for a three months period. The half-lives of some selected isomers ranged from 18 to 58 days. Whole-body autoradiography of blue mussels exposed to 14 C-labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) showed that the radioactivity was concentrated in the hepatopancreas with smaller amounts in the gills and foot.