A multi-biomarker study on Atlantic salmon (Salmo salar L.) affected by the emerging Red Skin Disease in the Baltic Sea.

For half a decade, the Atlantic salmon in the Baltic Sea has been facing severe health issues. Clinical signs like haemorrhage, erosions and ulcerative/necrotic skin conditions in returning adults have been reported from different Swedish rivers. These primary disease signs precede a secondary, terminal fungal infection. As initial investigations of the disease did not provide conclusive answers regarding the pathogenesis, this study was initiated to gain insight into a possible link between this so-called Red Skin Disease and anthropogenic influences. Therefore, returning salmon were caught in rivers along the Swedish coast and different tissues were sampled. The focus was put on the measurements of a battery of biomarkers as well as biochemical and haematological parameters, which were analysed using multivariate statistics. The main findings were a severe osmotic haemodilution, an immune response and an alteration of the carbohydrate metabolism in diseased fish. Furthermore, oxidative stress does not seem to be a likely factor in the pathogenesis. Concluding, certain changes in physiological parameters were shown to be indicative for the disease patterns, while others were ruled out as significant factors. Thus, this study contributes to the understanding of the Red Skin Disease and may act as a hypothesis generator for future studies.

Thyroid function and immune status in perch (Perca fluviatilis) from lakes contaminated with PFASs or PCBs.

The environment contains a multitude of man-made chemicals, some of which can act as endocrine disruptors (EDCs), while others can be immunotoxic. We evaluated thyroid disruption and immunotoxic effects in wild female perch (Perca fluviatilis) collected from two contaminated areas in Sweden; one site contaminated with per- and polyfluoroalkyl substances (PFASs) and two sites contaminated with polychlorinated biphenyls (PCBs), with one reference site included for each area. The hepatic mRNA expression of thyroid receptors α and ß, and the thyroid hormone metabolising iodothyronine deiodinases (dio1, dio2 and dio3) were measured using real-time PCR, while the levels of thyroid hormone T3 in plasma was analysed using a radioimmunoassay. In addition, lymphocytes, granulocytes, and thrombocytes were counted microscopically. Our results showed lower levels of T3 as well as lower amounts of lymphocytes and granulocytes in perch collected from the PFAS-contaminated site compared to reference sites. In addition, expressions of mRNA coding for thyroid hormone metabolising enzymes (dio2 and dio3) and thyroid receptor α (thra) were significantly different in these fish compared to their reference site. For perch collected at the two PCB-contaminated sites, there were no significant differences in T3 levels or in expression levels of the thyroid-related genes, compared to the reference fish. Fish from one of the PCB-contaminated sites had higher levels of thrombocytes compared with both the second PCB lake and their reference lake; hence PCBs are unlikely to be the cause of this effect. The current study suggests that lifelong exposure to PFASs could affect both the thyroid hormone status and immune defence of perch in the wild.

Assessing the effects of textile leachates in fish using multiple testing methods: From gene expression to behavior.

The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, children's bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

Rainbow Trout Maintain Intestinal Transport and Barrier Functions Following Exposure to Polystyrene Microplastics.

Ingestion has been proposed as a prominent exposure route for plastic debris in aquatic organisms, including fish. While the consequences of ingestion of large plastic litter are mostly understood, the impacts resulting from ingestion of microplastics (MPs) are largely unknown. We designed a study that aimed to assess impacts of MPs on fish intestinal physiology and examined integrity of extrinsic, physical and immunological barriers. Rainbow trout were exposed to polystyrene (PS) MPs (100-400 µm) via feed for a period of 4 weeks. Fish were fed four types of diets: control, diets containing virgin PS particles, or particles exposed to two different environmental matrices (sewage or harbor effluent). Extrinsic barrier disturbance in intestinal tissue was evaluated via histology. The paracellular permeability toward ions and molecules was examined using Ussing chambers and mRNA expression analysis of tight junction proteins. Active transport was monitored as transepithelial potential difference, short-circuits current and uptake rate of amino acid 3H-lysine. Immune status parameters were measured through mRNA expression level of cytokines, lysozyme activity, and hematological analysis of immune cells. We could not show that PS MPs induced inflammatory responses or acted as physical or chemical hazards upon ingestion. No measurable effects were exerted on fish intestinal permeability, active transport or electrophysiology.

Substances of emerging concern in Baltic Sea water: Review on methodological advances for the environmental assessment and proposal for future monitoring.

The Baltic Sea is among the most polluted seas worldwide. Anthropogenic contaminants are mainly introduced via riverine discharge and atmospheric deposition. Regional and international measures have successfully been employed to reduce concentrations of several legacy contaminants. However, current Baltic Sea monitoring programs do not address compounds of emerging concern. Hence, potentially harmful pharmaceuticals, UV filters, polar pesticides, estrogenic compounds, per- and polyfluoroalkyl substances, or naturally produced algal toxins are not taken into account during the assessment of the state of the Baltic Sea. Herein, we conducted literature searches based on systematic approaches and compiled reported data on these substances in Baltic Sea surface water and on methodological advances for sample processing and chemical as well as effect-based analysis of these analytically challenging marine pollutants. Finally, we provide recommendations for improvement of future contaminant and risk assessment in the Baltic Sea, which revolve around a combination of both chemical and effect-based analyses.

Bisphenol A and Bisphenol S Induce Endocrine and Chromosomal Alterations in Brown Trout.

Bisphenol A is a widely used compound found in large amount of consumer products. As concerns have been raised about its toxicological and public health effect, the use of alternatives to bisphenol A are now increasing. Bisphenol S is one of the analogues being used as a replacement for bisphenol A despite the fact that little is known about the effects of bisphenol S on living organisms. In this study, we investigated the potential endocrine and genotoxic effects of bisphenol A and bisphenol S in juvenile brown trout (Salmo trutta). The fish were exposed to the compounds for either 2 weeks or 8 weeks via sustained-release cholesterol implants containing doses of 2 mg/kg fish or 20 mg/kg fish of the substances. The effects on the thyroid hormone levels and the estrogenic disrupting marker vitellogenin were evaluated, along with the genotoxic markers micronucleated cells and erythrocyte nuclear abnormalities. An increase in plasma vitellogenin was observed in fish exposed to the high dose of bisphenol A for 2 weeks. At this experimental time the level of the thyroid hormone triiodothyronine (T3) in plasma was elevated after bisphenol S exposure at the high concentration, and paralleled by an increase of micronucleated cells. Moreover, bisphenol A induced an increase of micronuclei frequency in fish erythrocytes after the exposure at the lowest dose tested. Taken together the results indicate that both bisphenol A and its alternative bisphenol S cause endocrine disrupting and genotoxic effects in brown trout, although suggesting two different mechanisms of damage underlying bisphenol A and bisphenol S activity.

Naproxen affects multiple organs in fish but is still an environmentally better alternative to diclofenac.

The presence of diclofenac in the aquatic environment and the risks for aquatic wildlife, especially fish, have been raised in several studies. One way to manage risks without enforcing improved wastewater treatment would be to substitute diclofenac (when suitable from a clinical perspective) with another non-steroidal anti-inflammatory drug (NSAID) associated with less environmental risk. While there are many ecotoxicity-studies of different NSAIDs, they vary extensively in set-up, species studied, endpoints and reporting format, making direct comparisons difficult. We previously published a comprehensive study on the effects of diclofenac in the three-spined stickleback (Gasterosteus aculeatus). Our present aim was to generate relevant effect data for another NSAID (naproxen) using a very similar setup, which also allowed direct comparisons with diclofenac regarding hazards and risks. Sticklebacks were therefore exposed to naproxen in flow-through systems for 27 days. Triplicate aquaria with 20 fish per aquarium were used for each concentration (0, 18, 70, 299 or 1232 µg/L). We investigated bioconcentration, hepatic gene expression, jaw lesions, kidney and liver histology. On day 21, mortalities in the highest exposure concentration group unexpectedly reached ≥ 25 % in all three replicate aquaria, leading us to terminate and sample that group the same day. On the last day (day 27), the mortality was also significantly increased in the second highest exposure concentration group. Increased renal hematopoietic hyperplasia was observed in fish exposed to 299 and 1232 µg/L. This represents considerably higher concentrations than those expected in surface waters as a result of naproxen use. Such effects were observed already at 4.6 µg/L in the experiment with diclofenac (lowest tested concentration). Similar to the responses to diclofenac, a concentration-dependent increase in both relative hepatic gene expression of c7 (complement component 7) and jaw lesions were observed, again at concentrations considerably higher than expected in surface waters. Naproxen bioconcentrated less than diclofenac, in line with the observed effect data. An analysis of recent sales data and reported concentrations in treated sewage effluent in Sweden suggest that despite higher dosages used for naproxen, a complete substitution would only be expected to double naproxen emissions. In summary, naproxen and diclofenac produce highly similar effects in fish but the environmental hazards and risks are clearly lower for naproxen. Hence, if there are concerns for environmental risks to fish with diclofenac, a substitution would be advisable when naproxen presents an adequate alternative from a clinical point-of-view.

Characterization of the Zoarces viviparus liver transcriptome using massively parallel pyrosequencing.

BACKGROUND: The teleost Zoarces viviparus (eelpout) lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays. RESULTS: In the present study we present the first comprehensive characterization of the Zoarces viviparus liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of Gasterosteus aculeatus (stickleback) were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis. CONCLUSION: Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in Zoarces viviparus.

Microarray analysis of blood microvessels from PDGF-B and PDGF-Rbeta mutant mice identifies novel markers for brain pericytes.

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)-/- and PDGF beta receptor (PDGFRbeta)-/- mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B-/- and PDGFRbeta-/- microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.

Diclofenac affects kidney histology in the three-spined stickleback (Gasterosteus aculeatus) at low µg/L concentrations.

Diclofenac, a commonly used non-steroidal anti-inflammatory drug, is considered for regulation under the European water framework directive. This is because effects on fish have been reported at concentrations around those regularly found in treated sewage effluents (∼1µg/L). However, a recent publication reports no effects on fish at 320µg/L. In this study, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to 0, 4.6, 22, 82 and 271µg/L diclofenac in flow-through systems for 28days using triplicate aquaria per concentration. At the highest concentration, significant mortalities were observed already after 21days (no mortalities found up to 22µg/L). Histological analysis revealed a significant increase in the proportion of renal hematopoietic tissue (renal hematopoietic hyperplasia) after 28days at the lowest concentration and at all higher concentrations, following a clear dose-response pattern. Skin ulcerations of the jaw were noted by macroscopic observations, primarily at the two highest concentrations. No histological changes were observed in the liver. There was an increase in the relative hepatic mRNA levels of c7 (complement component 7), a gene involved in the innate immune system, at 22µg/L and at all higher concentrations, again following a clear dose-response. The bioconcentration factor was stable across concentrations, but lower than reported for rainbow trout, suggesting lower internal exposure to the drug in the stickleback. In conclusion, this study demonstrates that diclofenac causes histological changes in the three-spined stickleback at low µg/L concentrations, which cause concern for fish populations exposed to treated sewage effluents.

Biomarker responses in eelpouts from four coastal areas in Sweden, Denmark and Germany.

To increase our understanding of possible chemical impacts on coastal fish populations in the Baltic Sea, Kattegat and Skagerrak, the viviparous eelpout (Zoarces viviparus) was used as sentinel species in two major sampling campaigns (spring and autumn) in 16 different coastal sites. Condition factor (CF), liver somatic index (LSI), gonad somatic index (GSI) were measured and the activity of the hepatic enzymes ethoxyresorufin-O-deethylase (EROD), glutathione reductase GR), glutathione S-transferase (GST), catalase (CAT) and muscular activity of acetylcholinesterase (AChE) were assessed. PAH metabolites in bile were also analyzed. The most notable finding in the data set was the low EROD activity in eelpouts collected at the relatively polluted region in Germany compared to the other regions, which could be due to an inhibition of the CYP1A-system or to adaptation to chronic exposure of pollutants in this area. Additionally, low AChE activity was noted in the German region in the autumn campaign and low AChE activity detected in the Danish region in the spring campaign. These differences suggest possible season-specific differences in the use and release of AChE-inhibiting chemicals in the Danish and German regions. Clustering of biomarkers on site level indicated a relationship between CF and GSI and suggested that sites with a high CF contained eelpout that put a larger effort into their larvae development. Clustering of the oxidative stress markers GR, GST and CAT on the individual level reflected a possible coordinated regulation of these enzymes. Overall, the results support the importance of taking into account general regional differences and seasonal variation in biomarker activity when monitoring and assessing the effects of pollution. Despite the expected seasonal variation for most of the measured endpoint, several markers (GSI, EROD and CF) vary similarly between all selected sites in both spring and autumn. This suggests that the differences between sites for these endpoints are independent of season.

A gene to organism approach--assessing the impact of environmental pollution in eelpout (Zoarces viviparus) females and larvae.

A broad biomarker approach was applied to study the effects of marine pollution along the Swedish west coast using the teleost eelpout (Zoarces viviparus) as the sentinel species. Measurements were performed on different biological levels, from the molecular to the organismal, including measurements of messenger RNA (mRNA), proteins, cellular and tissue changes, and reproductive success. Results revealed that eelpout captured in Stenungsund had significantly higher hepatic ethoxyresorufin O-deethylase activity, high levels of both cytochrome P4501A and diablo homolog mRNA, and high prevalence of dead larvae and nuclear damage in erythrocytes. Eelpout collected in Göteborg harbor displayed extensive macrovesicular steatosis, whereby the majority of hepatocytes were affected throughout the liver, which could indicate an effect on lipid metabolism. Results also indicate that eelpouts collected at polluted sites might have an affected immune system, with lower mRNA expression of genes involved in the innate immune system and a higher number of lymphocytes. Biomarker assessment also was performed on livers dissected from unborn eelpout larvae collected from the ovary of the females. No significant differences were noted, which might indicate that the larvae to some extent are protected from effects of environmental pollutants. In conclusion, usage of the selected set of biological markers, covering responses from gene to organism, has demonstrated site-specific biomarker patterns that provided a broad and comprehensive picture of the impact of environmental stressors.

Hepatic transcriptome profiling indicates differential mRNA expression of apoptosis and immune related genes in eelpout (Zoarces viviparus) caught at Göteborg harbor, Sweden.

The physiology and reproductive performance of eelpout (Zoarces viviparus) have been monitored along the Swedish coast for more than three decades. In this study, transcriptomic profiling was applied for the first time as an exploratory tool to search for new potential candidate biomarkers and to investigate possible stress responses in fish collected from a chronically polluted area. An oligonucleotide microarray with more than 15,000 sequences was used to assess differentially expressed hepatic mRNA levels in female eelpout collected from the contaminated area at Göteborg harbor compared to fish from a national reference site, Fjällbacka. Genes involved in apoptosis and DNA damage (e.g., SMAC/diablo homolog and DDIT4/DNA-damage-inducible protein transcript 4) had higher mRNA expression levels in eelpout from the harbor compared to the reference site, whereas mRNA expression of genes involved in the innate immune system (e.g., complement components and hepcidin) and protein transport/folding (e.g., signal recognition particle and protein disulfide-isomerase) were expressed at lower levels. Gene Ontology enrichment analysis revealed that genes involved biological processes associated with protein folding, immune responses and complement activation were differentially expressed in the harbor eelpout compared to the reference site. The differential mRNA expression of selected genes involved in apoptosis/DNA damage and in the innate immune system was verified by quantitative PCR, using the same fish in addition to eelpout captured four years later. Thus, our approach has identified new potential biomarkers of pollutant exposure and has generated hypotheses on disturbed physiological processes in eelpout. Despite a higher mRNA expression of genes related to apoptosis (e.g., diablo homolog) in eelpout captured in the harbor there were no significant differences in the number of TUNEL-positive apoptotic cells between sites. The mRNA level of genes involved in apoptosis/DNA damage and the status of the innate immune system in fish species captured in polluted environments should be studied in more detail to lay the groundwork for future biomonitoring studies.

Physiology and mRNA expression in rainbow trout (Oncorhynchus mykiss) after long-term exposure to the new antifoulant medetomidine.

Medetomidine is under evaluation for use as an antifouling agent, and its effects on non-target aquatic organisms are therefore of interest. In this study, rainbow trout was exposed to low (0.5 and 5.0nM) concentrations of medetomidine for up to 54 days. Recently we have reported on effects on paleness and melanophore aggregation of medetomidine in these fish. Here, specific growth rates were investigated together with a broad set of physiological parameters including plasma levels of growth hormone (GH), insulin-like growth factor-I (IGF-I) and leptin, glucose and haemoglobin (Hb), hematocrit (Ht), condition factor, liver and heart somatic indexes (LSI, HSI). Hepatic enzyme activities of CYP1A (EROD activity), glutathione S-transferases (GST) and glutathione reductase (GR) were also measured. Additionally, hepatic mRNA expression was analysed through microarray and quantitative PCR in fish sampled after 31 days of exposure. Medetomidine at both concentrations significantly lowered blood glucose levels and the higher concentration significantly reduced the LSI. The mRNA expression analysis revealed few differentially expressed genes in the liver and the false discovery rate was high. Taken together, the results suggest that medetomidine at investigated concentrations could interfere with carbohydrate metabolism of exposed fish but without any clear consequences for growth.

Diclofenac in fish: blood plasma levels similar to human therapeutic levels affect global hepatic gene expression.

Diclofenac is a nonsteroidal anti-inflammatory drug frequently found in the aquatic environment. Previous studies have reported histological changes in the liver, kidney, and gills of fish at concentrations similar to those measured in treated sewage effluents (approximately 1 µg/L). Analyses or predictions of blood plasma levels in fish allow a direct comparison with human therapeutic plasma levels and may therefore be used to indicate a risk for pharmacological effects in fish. To relate internal exposure to a pharmacological interaction, we investigated global hepatic gene expression together with bioconcentration in blood plasma and liver of rainbow trout (Oncorhynchus mykiss) exposed to waterborne diclofenac. At the highest exposure concentration (81.5 µg/L), the fish plasma concentration reached approximately 88% of the human therapeutic levels (C(max) ) after two weeks. Using an oligonucleotide microarray followed by quantitative PCR, we found extensive effects on hepatic gene expression at this concentration, and some genes were found to be regulated down to the lowest exposure concentration tested (1.6 µg/L), corresponding to a plasma concentration approximately 1.5% of the human C(max) . Thus, at concentrations detected in European surface waters, diclofenac can affect the expression of multiple genes in exposed fish. Functional analysis of differentially expressed genes revealed effects on biological processes such as inflammation and the immune response, in agreement with the mode of action of diclofenac in mammals. In contrast to some previously reported results, the bioconcentration factor was found to be stable (4.02 ± 0.75 for blood plasma and 2.54 ± 0.36 for liver) regardless of the water concentration.

Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.

A new method for large scale isolation of kidney glomeruli from mice.

Here we report a new isolation method for mouse glomeruli. The method is fast and simple and allows for the isolation of virtually all glomeruli present in the adult mouse kidney with minimal contamination of nonglomerular cells. Mice were perfused through the heart with magnetic 4.5- micro m diameter Dynabeads. Kidneys were minced into small pieces, digested by collagenase, filtered, and collected using a magnet. The number of glomeruli retrieved from one adult mouse was 20,131 +/- 4699 (mean +/- SD, n = 14) with a purity of 97.5 +/- 1.7%. The isolated glomeruli retained intact morphology, as confirmed by light and electron microscopy, as well as intact mRNA integrity, as confirmed by Northern blot analysis. The method was applicable also to newborn mice, which allows for the isolation of immature developmental stage glomeruli. This method makes feasible transcript profiling and proteomic analysis of the developing, healthy and diseased mouse glomerulus.

The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells.

The entire cDNA corresponding to the C-terminal cysteine-rich domain of the human MUC2 apomucin, after the serine- and threonine-rich tandem repeat, was expressed in Chinese-hamster ovary-K1 cells and in the human colon carcinoma cell line, LS 174T. The C-terminus was expressed as a fusion protein with the green fluorescent protein and mycTag sequences and the murine immunoglobulin kappa-chain signal sequence to direct the protein to the secretory pathway. Pulse-chase studies showed a rapid conversion of the C-terminal monomer into a dimer in both Chinese-hamster ovary-K1 and LS 174T cells. Disulphide-bond-stabilized dimers secreted into the media of both cell lines had a higher apparent molecular mass compared with the intracellular forms. The MUC2 C-terminus was purified from the spent culture medium and visualized by molecular electron microscopy. The dimer nature of the molecule was visible clearly and revealed that each monomer was attached to the other by a large globular domain. Gold-labelled antibodies against the mycTag or green fluorescent protein revealed that these were localized to the ends opposite to the parts responsible for the dimerization. The C-terminus expressed in LS 174T cells formed heterodimers with the full-length wild-type MUC2, but not with the MUC5AC mucin, normally expressed in LS 174T cells. The homodimers of the MUC2 C-termini were secreted continuously from the LS 174T cells, but no wild-type MUC2 secretion has been observed from these cells. This suggests that the information for sorting the MUC2 mucin into the regulated secretory pathway in cells having this ability is present in parts other than the C-terminus of MUC2.