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.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

Estuarine fish health assessment: evidence of wastewater impacts based on nitrogen isotopes and histopathology

Sewage effluent is a powerful agent of ecological change in estuaries. While the effects of sewage pollution on water quality are usually well documented, biological responses of exposed organisms are not. We quantified health impacts in the form of pathological tissue changes across multiple organs in estuarine fish exposed to elevated levels of treated wastewater. Structural pathologies were compared in wild populations of four fish species from two subtropical estuaries on the east coast of Australia that differ substantially in the amount of direct wastewater loadings. Uptake of sewage-derived  nitrogen by fish was traced with stable nitrogen isotopes. Pathologies were common in the liver, spleen, gill, kidney and muscle tissues, and included granulomas, melanomacrophage aggregates, and multiple deformities of the gill epithelia. Tissue deformities were more frequent in fish exposed directly to wastewater discharges. Mullet (Valamugil georgii) were most affected, with only a single specimen free of pathologies in the sewage-impacted estuary. Similarly, in those fish that had structural abnormalities, more deformities were generally found in individuals from sites receiving sewage. These spatial contrasts in impaired fish health correspond to significantly enriched δ₁₅N values in fish muscle as a consequence of fish assimilating sewage-N. Overall, the pattern of lower health and enriched δ₁₅N values in fish from sewage-impacted areas suggests that organism health is lowered by sewage inputs to estuaries. Measurements of organism health are required to understand the effects of sewage on estuarine ecosystems, and histopathology of fishes is a powerful tool to achieve this.

ATPase activity, proton gradients and proton secretion inhibition during experiments with Sepia officinalis and Sepioteuthis lessoniana

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here, we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues; as in teleost fish, epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a Pco2 between 0.2 and 0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization, and vital dye-staining techniques. We found one group of cells that is recognized by concavalin A and MitoTracker, which also expresses Na+/H+ exchangers (NHE3) and Na+-K+-ATPase. Similar to findings obtained in teleosts, these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE-based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy, suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa, which were identified as powerful acid-base regulators during hypercapnic challenges, already exhibit strong acid-base regulatory abilities during embryogenesis.

Differential acid-base regulation in various gills of the green crab Carcinus maenas: Effects of elevated environmental pCO2

Euryhaline decapod crustaceans possess an efficient regulation apparatus located in the gill epithelia, providing a high adaptation potential to varying environmental abiotic conditions. Even though many studies focussed on the osmoregulatory capacity of the gills, acid-base regulatory mechanisms have obtained much less attention. In the present study, underlying principles and effects of elevated pCO2 on acid-base regulatory patterns were investigated in the green crab Carcinus maenas acclimated to diluted seawater. In gill perfusion experiments, all investigated gills 4-9 were observed to up-regulate the pH of the hemolymph by 0.1-0.2 units. Anterior gills, especially gill 4, were identified to be most efficient in the equivalent proton excretion rate. Ammonia excretion rates mirrored this pattern among gills, indicating a linkage between both processes. In specimen exposed to elevated pCO2 levels for at least 7 days, mimicking a future ocean scenario as predicted until the year 2300, hemolymph K+ and ammonia concentrations were significantly elevated, and an increased ammonia excretion rate was observed. A detailed quantitative gene expression analysis revealed that upon elevated pCO2 exposure, mRNA levels of transcripts hypothesized to be involved in ammonia and acid-base regulation (Rhesus-like protein, membrane-bound carbonic anhydrase, Na+/K+-ATPase) were affected predominantly in the non-osmoregulating anterior gills.

Phosphate absorption across multiple epithelia in the Pacific hagfish (Eptatretus stoutii)

Inorganic phosphate (Pi) is an essential nutrient for all organisms, but in seawater, Pi is a limiting nutrient. This study investigated the primary mechanisms of Pi uptake in Pacific hagfish (Eptatretus stoutii) using ex vivo physiological and molecular techniques. Hagfish were observed to have the capacity to absorb Pi from the environment into at least three epithelial surfaces: the intestine, skin, and gill. Pi uptake in all tissues was concentration dependent, and saturable Pi transport was observed in the skin and gill at <2.0 mmol/l Pi. Gill and intestinal Pi uptake was sodium dependent, but Pi uptake into the skin increased under low sodium conditions. Gill Pi transport exhibited an apparent affinity constant ∼0.23–0.6 mmol/l Pi. A complete sequence of a type II sodium phosphate cotransporter (Slc34a) was obtained from the hagfish gill. Phylogenetic analysis of the hagfish Slc34a transporter indicates that it is earlier diverging than, and/or ancestral to, the other identified vertebrate Slc34a transporters (Slc34a1, Slc34a2, and Slc34a3). With the use of RT-PCR, the hagfish Slc34a transcript was detected in the intestine, skin, gill, and kidney, suggesting that this may be the transporter involved in Pi uptake into multiple epithelia in the hagfish. This is the first measurement of Pi uptake across the gill or skin of any vertebrate animal and first sodium phosphate cotransporter identified in hagfish.

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na+/K+-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na+/K+-ATPase with the polyclonal antibody alpha (H-300) raised against the human alpha1-subunit of Na+/K+-ATPase. Na+/K+-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na+/K+-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na+/K+-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na+/K+-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 µmol/g FM/h) than in those of L. vulgaris (31.8 ± 3.3 µmol/g FM/h). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 µmol ATP/g FM/h, respectively. High concentrations of Na+/K+-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO2) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and β-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane.

Transcriptome analyses of amoebic gill disease-affected atlantic salmon (Salmo salar) tissues reveal localized host gene suppression

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (± SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response. © 2008 Springer Science+Business Media, LLC.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways. © 2008 Elsevier Ltd. All rights reserved.

Analysis, characterisation and expression of gill-expressed carbonic anhydrase genes in the freshwater crayfish Cherax quadricarinatus

Changes in water quality parameters such as pH and salinity can have a significant effect on productivity of aquaculture species. Similarly, relative osmotic pressure influences various physiological processes and regulates expression of a number of osmoregulatory genes. Among those, carbonic anhydrase (CA) plays a key role in systemic acid–base balance and ion regulation. Redclaw crayfish (Cherax quadricarinatus) are unique in their ability to thrive in environments with naturally varied pH levels, suggesting unique adaptation to pH stress. To date, however, no studies have focused on identification and characterisation of CA or other osmoregulatory genes in C. quadricarinatus. Here, we analysed the redclaw gill transcriptome and characterized CA genes along with a number of other key osmoregulatory genes that were identified in the transcriptome. We also examined patterns of gene expression of these CA genes when exposed to three pH treatments. In total, 72,382,710 paired end Illumina reads were assembled into 36,128 contigs with an average length of 800 bp. Approximately 37% of contigs received significant BLAST hits and 22% were assigned gene ontology terms. Three full length CA isoforms; cytoplasmic CA (ChqCAc), glycosyl-phosphatidylinositol-linked CA (ChqCAg), and β-CA (ChqCA-beta) as well as two partial CA gene sequences were identified. Both partial CA genes showed high similarity to ChqCAg and appeared to be duplicated from the ChqCAg. Full length coding sequences of Na+/K+-ATPase, V-type H+-ATPase, sarcoplasmic Ca+-ATPase, arginine kinase, calreticulin and Cl− channel protein 2 were also identified. Only the ChqCAc gene showed significant differences in expression across the three pH treatments. These data provide valuable information on the gill expressed CA genes and their expression patterns in freshwater crayfish. Overall our data suggest an important role for the ChqCAc gene in response to changes in pH and in systemic acid–base balance in freshwater crayfish.

Comparative analysis of gill transcriptomes of two freshwater crayfish, Cherax cainii and C. destructor

We undertook deep sequencing of gill transcriptomes from two freshwater crayfish, Cherax cainii and Cherax destructor, in order to generate genomic resources for future genomics research. Over 83 and 100 million high quality (quality score (Q) ≥ 30) paired-end Illumina reads (150 bp) were assembled into 147,101 and 136,622 contigs in C. cainii and C. destructor, respectively. A total of 24,630 and 23,623 contigs received significant BLASTx hits and allowed the identification of multiple gill expressed candidate genes associated with pH and salinity balance. These functionally annotated transcripts will provide a resource to facilitate comparative genomic research in the genus Cherax, and in particular allow insights into respiratory and osmoregulatory physiology of this group of animals.

Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus

The pH and salinity balance mechanisms of crayfish are controlled by a set of transport-related genes. We identified a set of the genes from the gill transcriptome from a freshwater crayfish Cherax quadricarinatus using the Illumina NGS-sequencing technology. We identified and characterized carbonic anhydrase (CA) genes and some other key genes involved in systematic acid-base balance and osmotic/ionic regulation. We also examined expression patterns of some of these genes across different sublethal pH levels [1]. A total of 72,382,710 paired-end Illumina reads were assembled into 36,128 contigs with an average length of 800 bp. About 37% of the contigs received significant BLAST hits and 22% were assigned gene ontology terms. These data will assist in further physiological-genomic studies in crayfish.

Cyclic nucleotide signaling in intestinal epithelia: getting to the gut of the matter

The intestine is the primary site of nutrient absorption, fluid-ion secretion, and home to trillions of symbiotic microbiota. The high turnover of the intestinal epithelia also renders it susceptible to neoplastic growth. These diverse processes are carefully regulated by an intricate signaling network. Among the myriad molecules involved in intestinal epithelial cell homeostasis are the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP). These cyclic nucleotides are synthesized by nucleotidyl cyclases whose activities are regulated by extrinsic and intrinsic cues. Downstream effectors of cAMP and cGMP include protein kinases, cyclic nucleotide gated ion channels, and transcription factors, which modulate key processes such as ion-balance, immune response, and cell proliferation. The web of interaction involving the major signaling pathways of cAMP and cGMP in the intestinal epithelial cell, and possible cross-talk among the pathways, are highlighted in this review. Deregulation of these pathways occurs during infection by pathogens, intestinal inflammation, and cancer. Thus, an appreciation of the importance of cyclic nucleotide signaling in the intestine furthers our understanding of bowel disease, thereby aiding in the development of therapeutic approaches.