Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia

In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3 h, and was maximal after 6 h of exposure to TCDD, B [alp and Aroclor 1254. The estimated 6 h EC50 for TCDD, B [a]P, and Aroclor 1254 was 1.2x10(-9), 5.7x10(-8) and 6.6x10(-6) M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24 h after exposure. The estimated 24 It EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4x10(-9), 8.1x10(-8) and 7.3x10(-6) M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants. (c) 2006 Elsevier B.V. All rights reserved.

EROD activities in a primary cell culture of grass carp (Ctenopharyngodon idellus) hepatocytes exposed to polychlorinated aromatic hydrocarbonas

Aryl hydrocarbon (Ah) receptor (Ah-agonist) effects of environmental samples containing polychlorinated aromatic hydrocarbons were evaluated using a 7-ethoxyresorufin-O-deethylase (FROD) assay of a primary hepatocyte culture from grass carp (Ctenopharyngodon idellus). The results were compared with those obtained from the assay using the rat hepatoma cell line H4IIE and chemical analysis using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). A dose-response relationship was observed between the EROD activities, either from primary hepatocyte culture assay or from H4IIE assay, and concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that the assay based on the H4IIE cell line (EC50 = 0.83 mug/mL) is more sensitive to TCDD than the assay based on primary hepatocyte Culture (EC50 = 9.7 pg/mL). In tests of environmental samples, the results from the assay using primary hepatocyte culture were comparable to those from the assay using the H4IIE cell line and chemical analysis of concentrations of mixtures of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDF). The lack of a change in the activities of glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) in cell culture upon exposure to TCDD indirectly indicates that the compound is persistent to biodegradation in the cell culture system. (C) 2004 Elsevier Inc. All rights reserved.

Antibody response of carp, Cyprinus carpio to the cestode, Bothriocephalus acheilognathi

The humoral antibody response and the number of pronephric antibody-secreting cells were examined in naturally Bothriocephalus acheilognathi-infected carp. Cyprinus carpio, and in those injected intraperitoneally with an extract of the cestode. In the extract-injected fish, specific antibody was detected 3 weeks after a second injection given 2 weeks after the primary injection, and antibody levels persisted for more than 200 days. A third injection also enhanced the antibody level in the extract-injected carp. The numbers of antibody-secreting cells were significantly higher in carp injected 3 times with the extract than in the control. In naturally-infected fish, the serum antibody levels and the number of pronephric antibody-secreting cells were higher in infected fish than in uninfected individuals although this difference was not statistically significant. The relevance of these results to immune protection against infection is discussed.

Response of coastal marine eco-environment to river fluxes into the sea: A case study of the Huanghe (Yellow) River mouth and adjacent waters

The impact of the Huanghe (Yellow) River outflows on its estuary was investigated with river gauging and shipboard hydrographic observations. The river flux has been decreasing dramatically; the discharges of water and sediment in the 1990s dropped to 27.4% and 31.9% of those in the 1950s, respectively, resulting in frequent and lengthy events of downstream channel dry-up since the 1970s. There were accumulatively 897 zero-flow days during the 1990s in the river course below the Lijin Hydrological Station, 100 km upstream from the river mouth, which is 82.4% of that in 1972. As freshwater input decreases, river-borne nutrients to the estuarine increased significantly. Concentration of dissolved inorganic nitrogen (DIN) in the 1990s was four times of that in 1950s. Changes in amount and content of the riverine inputs have greatly affected the estuarine ecosystem. Over the past several decades, sea surface temperature and salinity in the estuary and its adjacent waters increased and their distribution pattern altered in response to the reduction of freshwater inflow. The distribution of and seasonal succession in nutrient concentrations in the surface layer have also changed with a shift of river outlet and the decrease in riverine nutrient loads. Furthermore, deterioration of estuarine ecosystem by less river input has decreased primary productivity in the deltaic region waters, and in turn depressed the fishery. (C) 2008 Published by Elsevier Ltd.

Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve

Marine sponge cell culture is a potential route for the sustainable production of sponge-derived bioproducts. Development of a basal culture medium is a prerequisite for the attachment, spreading, and growth of sponge cells in vitro. With the limited knowledge available on nutrient requirements for sponge cells, a series of statistical experimental designs has been employed to screen and optimize the critical nutrient components including inorganic salts (ferric ion, zinc ion, silicate, and NaCl), amino acids (glycine, glutamine, and aspartic acid), sugars (glucose, sorbitol, and sodium pyruvate), vitamin C, and mammalian cell medium (DMEM and RPMI 1640) using MTT assay in 96-well plates. The marine sponge Hymeniacidon perleve was used as a model system. Plackett-Burman design was used for the initial screening, which identified the significant factors of ferric ion, NaCl, and vitamin C. These three factors were selected for further optimization by Uniform Design and Response Surface Methodology (RSM), respectively. A basal medium was finally established, which supported an over 100% increase in viability of sponge cells.