Effects of Dietary Copper and Escherichia coli Challenge on the Immune Response and Gill Oxidative Balance in the Freshwater Mussel Diplodon chilensis.

Copper is a water and sediment pollutant that can be biomagnified by phytoplankton, and it often co-occurs with fecal bacteria. We addressed the combined effects of copper and Escherichia coli on the immune response and gill oxidative balance of the freshwater mussel Diplodon chilensis. Bivalves were sorted into four groups fed with 1) control algae, 2) bacteria (E. coli), 3) copper-enriched algae (Cu2+ ) algae, and 4) copper-enriched algae followed by bacteria (Cu2+ + E. coli). Cellular and humoral immune and cytotoxic variables were analyzed in hemolymph, and detoxifying/antioxidant enzyme activities (glutathione S-transferase [GST] and catalase [CAT]) and lipid peroxidation (thiobarbituric acid reactive substances [TBARS]) were studied in gill tissue. The total hemocyte number increased after Cu2+ exposure, independently of the E. coli challenge. The proportion of hyalinocytes significantly diminished in the E. coli and Cu2+ groups but not in Cu2+ + E. coli groups; granulocytes significantly increased with E. coli but not with Cu2+ + E. coli treatments. Phagocytic activity was higher in all treatments than in control mussels. Acid phosphatase activity was increased by E. coli and inhibited by Cu2+ and Cu2+ + E. coli. Both E. coli and Cu2+ but not Cu2+ + E. coli augmented alkaline phosphatase activity. The Cu2+ and Cu2+ + E. coli treatments reduced the lysosomal membrane stability and cell viability. Humoral bacteriolytic and phenol oxidase activities were not affected by any treatment. The Cu2+ treatment induced gill CAT and GST activities and increased TBARS levels. The Cu2+ + E. coli treatment reversed this CAT and GST stimulation and increased the Cu2+ effect on TBARS. Dietary Cu2+ affects bivalves' immunological and oxidative status and impairs defensive responses against bacteria. In turn, E. coli potentiates the gill oxidative effects of Cu2+ . Environ Toxicol Chem 2023;42:154-165. © 2022 SETAC.

Microcystin-LR modulates multixenobiotic resistance proteins in the middle intestine of rainbow trout, Oncorhynchus mykiss.

Global climate change favors explosive population growth events (blooms) of phytoplanktonic species, often producing toxic products, e.g., several genera of cyanobacteria synthesize a family of cyanotoxins called microcystins (MCs). Freshwater fish such as the rainbow trout Oncorhynchus mykiss can uptake MCs accumulated in the food chain. We studied the toxic effects and modulation of the activity and expression of multixenobiotic resistance proteins (ABCC transporters and the enzyme glutathione S-transferase (GST) in the O. mykiss middle intestine by microcystin-LR (MCLR). Juvenile fish were fed with MCLR incorporated in the food every 12 h and euthanized at 12, 24, or 48 h. We estimated the ABCC-mediated transport in ex vivo intestinal strips to estimate ABCC-mediated transport activity. We measured total and reduced (GSH) glutathione contents and GST and glutathione reductase (GR) activities. We studied MCLR cytotoxicity by measuring protein phosphatase 1 (PP1) activity and lysosomal membrane stability. Finally, we examined the relationship between ROS production and lysosomal membrane stability through in vitro experiments. Dietary MCLR had a time-dependent effect on ABCC-mediated transport, from inhibition at 12 h to a significant increase after 48 h. GST activity decreased only at 12 h, and GR activity only increased at 48 h. There were no effects on GSH or total glutathione contents. MCLR inhibited PP1 activity and diminished the lysosomal membrane stability at the three experimental times. In the in vitro study, the lysosomal membrane stability decreased in a concentration-dependent fashion from 0 to 5 µmol L - 1 MCLR, while ROS production increased only at 5 µmol L - 1 MCLR. MCLR did not affect mRNA expression of abcc2 or gst-π. We conclude that MCLR modulates ABCC-mediated transport activity in O. mykiss's middle intestine in a time-dependent manner. The transport rate increase does not impair MCLR cytotoxic effects.

Effects of paralytic shellfish toxins on the middle intestine of Oncorhynchus mykiss: Glutathione metabolism, oxidative status, lysosomal function and ATP-binding cassette class C (ABCC) proteins activity.

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

Alterations in the intestine of Patagonian silverside (Odontesthes hatcheri) exposed to microcystin-LR: Changes in the glycosylation pattern of the intestinal wall and inhibition of multidrug resistance proteins efflux activity.

Accumulation and toxicity of cyanobacterial toxins, particularly microcystin-LR (MCLR) have been extensively studied in fish and aquatic invertebrates. However, MCLR excretion mechanisms, which could reduce this toxin's effects, have received little attention. The Patagonian silverside, Odontesthes hatcheri, is an omnivorous-planktivorous edible fish, which has been shown to digest cyanobacterial cells absorbing MCLR and eliminating the toxin within 48h without suffering significant toxic effects. We studied the effects of MCLR on glycoconjugate composition and the possible role of multidrug resistance associated proteins (Abcc) in MCLR export from the cells in O. hatcheri intestine. We treated O. hatcheri with 5µg MCLRg(-1) body mass administered with the food. Twenty four hours later, the intestines of treated and control fish were processed for lectin-histochemistry using concanavalin A (ConA), Triticum vulgaris agglutinin (WGA), and Dolichos biflorus agglutinin (DBA). MCLR affected the distribution of glycoconjugates by augmenting the proportion of ConA-positive at the expense of WGA-positive cells. We studied MCLR effects on the transport of the Abcc-like substrates 2,4-dinitrophenyl-S-glutathione (DNP-SG) and calcein in ex vivo intestine preparations (everted and no-everted sacs and strips). In treated preparations, CDNB together with MCLR (113µg MCLRg(-1) intestine, equivalent to 1.14µmolL(-1) when applied in the bath) or the Abcc inhibitor, MK571 was applied for one hour, during which DNP-SG was measured in the bath every 10min in order to calculate mass-specific DNP-SG transport rate. MCLR significantly inhibited DNP-SG transport (p<0.05), especially in middle intestine (47 and 24%, for luminal and serosal transport, respectively). In middle intestine strips, MCLR and MK571inhibited DNP-SG transport in a concentration dependent fashion (IC50 3.3 and 0.6µmolL(-1), respectively). In middle intestine strips incubated with calcein-AM (0.25µmolL(-1)), calcein efflux was inhibited by MCLR (2.3µmolL(-1)) and MK571 (3µmolL(-1)) by 38 and 27%, respectively (p<0.05). Finally, middle intestine segments were incubated with different concentrations of MCLR applied alone or together with 3µM MK571. After one hour, protein phosphatase 1 (PP1) activity, the main target of MCLR, was measured. 2.5µM MCLR did not produce any significant effect, while the same amount plus MK571 inhibited PP1 activity (p<0.05). This effect was similar to that of 5µM MCLR. Our results suggest that in O. hatcheri enterocytes MCLR is conjugated with GSH via GST and then exported to the intestinal lumen through Abcc-like transporters. This mechanism would protect the cell from MCLR toxicity, limiting toxin transport into the blood, which is probably mediated by basolateral Abccs. From an ecotoxicological point of view, elimination of MCLR through this mechanism would reduce the amount of toxin available for trophic transference.

Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D. chilensis, continuous and long term feeding with E. gracilis cells favors immune response and reduces the damage caused by sewage pollution exposure on hemocytes. Nevertheless, diet and transplantation procedures may produce negative effects on the oxidative balance of gills and digestive gland and should be taken into account for bioremediation strategies.

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Biomarker responses to sewage pollution in freshwater mussels (Diplodon chilensis) transplanted to a Patagonian river.

Field and laboratory experiments were combined to evaluate biomarker responses of Diplodon chilensis to sewage pollution. Mussels from an unpolluted area in Lacar lake (S0) were caged at a reference site (S1) and at two sites with increasing sewage pollution (S2, S3) in Pocahullo river (all in Argentina). After 1 month, gill (g) glutathione S-transferase (GST) and catalase (CAT) activities, and lipid peroxidation (TBARS) were found to be significantly elevated in S3, gGST being positively correlated with fecal bacteria (FC) concentration. Digestive gland (dg) enzyme activities were depressed and dgTBARS were increased in all transplanted mussels. After 3 mo, most variables returned to control levels in S1 mussels except for dgCAT and dgTBARS. After seven months, GST and CAT activities of S0 and S3 mussels were evaluated in the laboratory, before and after acute exposure (8 h) to high fecal bacteria concentration ([FC] in S3x 2). gGST increased in both groups, while dgGST responded only in S3 mussels. gCAT and dgCAT activities were similarly increased by acute exposure in both groups. Our results suggest that gGST and gCAT are suitable biomarkers for high FC pollution regardless of previous exposure history. In addition, we show that dgCAT is sensitive to the acute increase in FC load, both in naive and long-term exposed individuals, while dgGST becomes responsive after long-term acclimatization.

Health status and bioremediation capacity of wild freshwater mussels (Diplodon chilensis) exposed to sewage water pollution in a glacial Patagonian lake.

Deleterious effects on health and fitness are expected in mussels chronically exposed to sewage water pollution. Diplodon chilensis inhabiting SMA, an area affected by untreated and treated sewage water, shows increased hemocyte number and phagocytic activity, while bacteriolytic and phenoloxidase activities in plasma and reactive oxygen species production in hemocytes are lower compared to mussels from an unpolluted area (Yuco). There are not differences in cell viability, lysosomal membrane stability, lipid peroxidation and total oxygen scavenging capacity between SMA and Yuco mussels' hemocytes. Energetic reserves and digestive gland mass do not show differences between groups; although the condition factor is higher in SMA than in Yuco mussels. Gills of SMA mussels show an increase in mass and micronuclei frequency compared to those of Yuco. Mussels from both sites reduce bacterial loads in polluted water and sediments, improving their quality with similar feeding performance. These findings suggest that mussels exposed to sewage pollution modulate physiological responses by long-term exposure; although, gills are sensitive to these conditions and suffer chronic damage. Bioremediation potential found in D. chilensis widens the field of work for remediation of sewage bacterial pollution in water and sediments by filtering bivalves.

Accumulation and biochemical effects of microcystin-LR on the Patagonian pejerrey (Odontesthes hatcheri) fed with the toxic cyanobacteria Microcystis aeruginosa.

We studied accumulation and biochemical effects of microcystin-LR (MCLR) in Odontesthes hatcheri after dietary administration of the cyanobacteria Microcystis aeruginosa (1.3 µg MCLR/g body mass, incorporated in standard fish food). After 12 h, MCLR content in liver did not differ between fish fed with crushed or intact cells, demonstrating O. hatcheri's capacity to digest cyanobacteria and absorb MCLR. In the second experiment, fish received toxic cells, non-toxic cells, or control food; MCLR accumulation was monitored for 48 h. Protein phosphatase 1 (PP1), catalase (CAT), glutathione-S-transferase (GST) activities, and lipid peroxidation (as MDA) were measured in liver and intestine. Methanol-extractable MCLR was determined by PP1 inhibition assay (PPIA); extractable and protein-bound MCLR were measured by Lemieux oxidation-gas chromatography/mass spectrometry (GC/MS). MCLR accumulated rapidly up to 22.9 and 9.4 µg MCLR/g in intestine and liver, respectively, followed by a decreasing tendency. Protein-bound MCLR represented 66 to ca. 100 % of total MCLR in both tissues. PP1 activity remained unchanged in intestine but was increased in liver of MCLR treated fish.CAT and GST activities and MDA content were significantly increased by MCLR only in liver. We conclude that O. hatcheri is able to digest cyanobacteria, accumulating MCLR mostly bound to proteins. Our data suggest that this freshwater fish can be adversely affected by cyanobacterial blooms. However, the rapid decrease of the detectable MCLR in both tissues could imply that sublethal toxin accumulation is rapidly reversed.