The cerium oxide nanoparticles toxicity induced physiological, histological and biochemical alterations in freshwater mussels, Unio crassus.

INTRODUCTION: Releasing of cerium oxide nanoparticles (nano-CeO2) to the nature has increased due to the widespread use in many fields ranging from cosmetics to the food industry. Therefore, nano-CeO2 has been included in the Organization for Economic Co-operation and Development's (OECD) priority list for engineering nanomaterials. In this study, the effects of nano-CeO2 on the freshwater mussels were investigated to reveal the impact on the freshwater systems on model organism. METHODS: First, the chemical and structural properties of nano-CeO2 were characterized in details. Second, the freshwater mussels were exposed to environmentally relevant concentrations of nano-CeO2 as 10 mg, 25 mg and 50 mg/L during 48-h and 7-d. Third, after the exposure periods, hemolymph and tissue samples were taken to analyse the Total Hemocyte Counts (THCs) histology and oxidative stress parameters (total antioxidant status, glutathione, glutathione-S-transferase, and advanced oxidative protein products). RESULTS: Significant decrease of the THCs was observed in the nano-CeO2 exposed mussels compared to the control group (P < 0.05). The histological results showed a positive association between nano-CeO2 exposure concentration in the water and level of tissue damage and histopathological alterations were detected in the gill and the digestive gland tissues. Oxidative stress parameters were slightly affected after exposure to nano-CeO2 (P > 0.05). In conclusion, this study showed that acute exposure of freshwater mussels to nano-CeO2 did not pose significant biological risk. However, it has been proven that mussels are able to accumulate nano-CeO2 significantly in their bodies. CONCLUSION: This suggests that nano-CeO2 may be a potential risk to other organisms in the ecosystem through trophic transfer in the food-web based on their habitat and niche in the ecosystem.

Pancreatic cancer, depression, and spirituality in therapy: "Unio Mystica" and "Achrayut," 2 case reports.

OBJECTIVES: Pancreatic cancer is a major site of gastrointestinal tumors and remains a leading cause of cancer death in adults in the United States. There is also a strong association between pancreatic cancer and depression. When struggling with cancer, along the different phases of illness, a human being is confronted with manifold issues, which might profoundly interfere with their sense of meaning and purpose. METHODS: From this standpoint, several different therapeutic techniques have been designed to manage the psychological needs of the patients. Here we provide 2 clinical scenarios, where there was a strong religious correlation to the therapeutic techniques employed with patients suffering from pancreatic cancer. RESULTS: The 2 cases described showed some improvement in their overall life view and could recalibrate their expectations based on a strong religious foundation. SIGNIFICANCE OF RESULTS: The role of religion and spirituality in health has also received increasing attention in literature. Religion and spirituality can help patients with cancer find meaning in their illness, provide comfort in the face of existential fears, and receive support from a community of like-minded individuals. In effect, they also provide evidence toward the scope of and integrating the domain of spirituality into holistic cancer care.

Combined effect of microplastic, salinomycin and heating on Unio tumidus.

Microplastic (MP) and heating (T) suspected to modulate biological effects of aquatic contaminants. Salinomycin (Sal) is veterinary antibiotic and anticancer agent. The goal of this study was to examine the multistress effect of MP, Sal and T on the bioindicator bivalve mollusc. The Unio tumidus were treated with MP (1 mg L-1), Sal (0.6 µg L-1), their combination under 18° C (Mix) and 25° C (MixT) for 14 days. The digestive glands were analyzed. MP and Sal did not cause changes of Mn- and Cu,Zn-SOD, lipid peroxidation and Cyp-450-depended EROD levels, whereas catalase, GST and protein carbonyls (Sal-group) increased compared to control. In the Mix-group, enzymes, particularly EROD and GST (by 34% and 115% respectively) were up-regulated. However, in the MixT-group, they were corresponding to control or lesser (EROD, catalase). Our findings emphasize the need to take into account multistress interactions in the MP environmental risk assessment.

The impact of iron coagulant on the behavior and biochemistry of freshwater mussels Anodonta cygnea and Unio tumidus during lake restoration.

Iron (Fe) treatment is one of the most commonly used methods to restore eutrophic lakes and reservoirs. The Fe-based coagulants dosage results in an almost immediate improvement in water quality at a relatively low cost. However, the effects of the application of coagulants are not always predictable, and the scale of the risks is not fully understood. The dosage of coagulants changes the chemical and physical properties of water, thereby affecting aquatic biocenoses. In this study, several laboratory experiments were conducted to evaluate the effects of Fe-based coagulant dosage on two bivalves species: Anodonta anatina and Unio tumidus. Their ability to efficiently filter water and reduce seston makes them a key component of aquatic ecosystems in terms of maintaining proper ecological health and stable functioning. Behavioral response, biochemical parameters, and body chemistry changes in mussels exposed to different doses of coagulant were surveyed. A dose-dependent reduction in filtration activity of both species was observed. As early as 10 g Fe m2 (which is a moderate dose used in lakes restoration), mussels of both species almost completely reduced their filtration activity and remained with closed valves for several subsequent days. Significant Fe accumulation in muscles of bivalves exposed to coagulant was also observed. This was particularly the case when very high doses of coagulant were applied. Then, the iron content in leg muscles of both species increased over fourfold. At the same time, a decrease in muscles calcium and phosphorus content was observed. No symptoms of oxidative stress (TBARS, H2O2) after mussels exposure to coagulants were found. The results suggest that the application of Fe-based coagulant for water ecosystem restoration may be a threat to the mussels population. These findings are significant for decisions on the selection of restoration methods for a specific lake.

Characterization of ATPases in the gill of freshwater mussel (Unio tigridis) and effects of ionic and nanoparticle forms of aluminium and copper.

Mussels are often used to monitor the aquatic systems for different ecological aims, as they are one of the best bioindicator animals. However, the characterization of biomarkers should be known before using them in environmental monitoring and toxicology studies. There is no study to our knowledge on the characterization of Na-ATPase and Ca-ATPase in tissues of freshwater mussel (Unio tigridis). Thus, this study was undertaken to characterize the optimum working conditions of Na-ATPase and Ca-ATPase in the gill of mussels, determining the highest levels of parameters (Na+, Mg2+, Ca2+, ATP, pH, temperature, enzyme amount, incubation time) to obtain maximum activity. The present study also aimed to investigate in vitro effects of ionic and nanoparticle (Al2O3, CuO) forms of aluminium and copper (0, 30, 90, 270 µg/L) on the activities of Na-ATPase and Ca-ATPase. Data showed that there was no ouabain-sensitive ATPase activity in the gill up to 10 mM ouabain concentrations. Na-ATPase and Ca-ATPase activities in the gill of control mussels were 5.124 ± 0.373 and 3.750 ± 0.211 µmol Pi/mg pro./h, respectively. Exposure to different concentrations of nanoparticles did not alter significantly (P > 0.05) the activities of Na-ATPase and Ca-ATPase in vitro, whereas the same concentrations of ionic aluminium and copper significantly decreased (P < 0.05) the enzyme activities. Data emphasized that there were different modes of action between ionic and nanoparticle forms of aluminium and copper. Data also suggested that in vivo studies should also be carried out to estimate better the effects of nanoparticle and ionic forms of metals on ATPases of U. tigridis.

Biochemical and molecular alterations in freshwater mollusks as biomarkers for petroleum product, domestic heating oil.

To investigate the effect one of the oil products, domestic heating oil (DHO), on freshwater mollusks, Unio tigridis and Viviparous bengalensis were exposed to three DHO concentrations for each species (5.8, 8.7, and 17.4 ml L-1 for mussels; 6.5, 9.7, and 19.5 mlL-1 for snails, respectively). Antioxidant enzymes (superoxide dismutase, catalase), malondialdehyde, acetylcholinesterase and DNA damage in both species tissues were monitored over 21 days. The results showed that both antioxidant enzymes concentration (SOD and CAT) increased in the lowest DHO concentrations (5.8, and 8.7 ml L-1), and then decreased in the highest concentration (17.4 ml L-1) as the same pattern for Unio tigridis, but this not occurred for Viviparous bengalensis. MDA values recorded significantly increased compared to control. No reduction was observed in AChE concentrations in soft tissues of both mollusks may due to that DHO was a non-neurotoxicant to Unio tigridis and Viviparous bengalensis. The results of DNA damage parameters were showed significant differences (p≤ 0.05) between control and DHO concentrations except lowest concentration for each parameter measured in digestive gland of Unio tigridis. As well as, these significant differences were recorded between control and three concentrations of DHO exposure for comet length, and tail length parameters, and between control and highest oil concentration for tail moment in Viviparous bengalensis. DHO has the ability to prevent the reproduction of Viviparous bengalensis snail relation to control, that is what we considered strong evidence of the toxicity properties of DHO on the reproductive status of this species of snails. SOD, CAT, and MDA were useful biomarkers for evaluating the toxicity of DHO in mussel and snails, and comet assay was a good tool to assess the potential genotoxicity of DHO.

A field study of hemolymph yolk protein levels in a bivalve (Unio tumidus) and future considerations for bivalve yolk protein as endocrine biomarker.

Induction of yolk protein in male fish is a recognized biomarker for estrogenic exposure because the estrogen-dependent induction mechanism is well investigated and there is a clear difference in yolk protein levels of unexposed males and females. Attempts have been made to use induction of bivalve yolk protein as biomarker for estrogenic exposure. However, several biomarker validation criteria have not yet been investigated e.g. an in-depth understanding of the induction mechanism and background variability is needed and reliable detection assays are yet to be developed. To obtain background knowledge about yolk protein levels freshwater bivalves (Unio tumidus) were collected in an uncontaminated Danish lake over the course of a year (33 collection dates). The hemolymph yolk protein concentration of 569 individuals was determined by a species specific enzyme-linked immunosorbent assay (ELISA) and male and female gonadal development cycles were established. The average yolk protein levels of males and females collected at each sampling date overlapped in some periods; the male and female range was 66,946 - 169,692 ng/mL and 88,731 - 681,667 ng/mL, respectively. Because male and female hemolymph yolk protein levels overlap, great care should be taken if yolk protein induction in bivalve hemolymph is considered as endocrine biomarker.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; comparison of the alkali-labile phosphate method (ALP) and a species-specific immunoassay (ELISA).

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17ß-estradiol (57, 164 and 512 ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus. The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 µM) with 0.4 µM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 µM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

Cadmium availability to freshwater mussel (Unio tumidus) in the presence of organic matter and UV radiation.

The influence of ultraviolet radiation (UV) on cadmium availability to freshwater mussel (Unio tumidus) in the presence of organic matter was investigated. Water solutions containing 1000, 500, and 250 microg x L(-1) of cadmium and 28.8 mg x L(-1) of total organic carbon (TOC) were exposed to ecologically relevant biologically effective UV-A and UV-B irradiances of 1.73 x 10(-6) W x cm(-2) and 1.50 x 10(-5) W x cm(-2) respectively for 12 h. The availability of cadmium was measured directly as Cd concentration in gills, mantle, digestive gland and foot, and indirectly as its impact on the content of essential cations. The concentration of cadmium ions, soluble forms of cadmium, pH and conductivity in UV irradiated solutions were also measured. UV exposures resulted in a decrease of the TOC to 25.9 mg x L(-1), followed by a decrease in the total concentration of Cd soluble forms, increase in the concentration of cadmium bound to particulate matter, and increase in the concentration of free cadmium ions in comparison to non-irradiated mixtures. Mussels from mixtures exposed to UV accumulated more cadmium than specimens from non-irradiated mixtures. This was confirmed by the fluctuations of essential cations, i.e. decreases in the contents of Zn, Ca, Fe and increase of the Cu content. Na/K molar ratios in gills were negatively correlated with cadmium content, while a positive trend occurred in other organs. We suggest that in UV-exposed water more ionic forms of cadmium occur. On the other hand more cadmium is also bound to particulate matter. Our research indicate that the increase of cadmium accumulation in mussels grown in humic acid solutions exposed to UV were most probably related to the increase in the concentration of cadmium bound to particulate matter.

A fucose-containing O-glycoepitope on bovine and human nucleolin.

In this paper, we demonstrate the existence and localization of fucosyl-containing O-glycoforms of nucleolin in cultured bovine endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody raised against gp273, a glycoprotein ligand for the sperm-egg interaction in the mollusc bivalve Unio elongatulus. The function and immunological properties of gp273 mainly depend on clustered Lewis-like, fucose-containing O-glycans. Here an anti-gp273 antibody was used to evaluate whether glycoepitopes similar to those of gp273 are part of potential ligands of selectins in endothelial cells. We found that anti-gp273 strongly and exclusively interacted with a 110 kDa protein in CVEC and A431 tumor cells. After partial purification, mass spectrometry identified the protein as nucleolin. This was confirmed by comparing anti-gp273 and anti-nucleolin antibody immunoblotting after nucleolin depletion. We confirmed that anti-gp273 binding to nuclear and extranuclear nucleolin was against a fucose-containing O-glycoepitope by immunoblot analysis of the protein after chemically removing O-glycans and by lectin-blot analysis of control and nucleolin-depleted samples. Using anti-gp273 IgG, we detected nucleolin on the plasma membrane and cytoplasm. O-Glycosylation may regulate the plethora of functions in which nucleolin is involved.

Effects of deltamethrin on antioxidant status and oxidative stress biomarkers in freshwater mussel, Unio elongatulus eucirrus.

In this study, the effects of deltamethrin on antioxidant status and oxidative stress biomarkers in digestive gland and gill of freshwater mussel, Unio elongatulus eucirrus, was examined. Deltamethrin was applied at concentrations of 25, 50, 100, 200, 400, 800 and 1,600 microg L(-1), for 1, 24, 48, 72 and 96 h. With increasing deltamethrin concentrations, the mussels exposed duration 1-96 h significantly increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione and catalase activity in digestive gland and gill of freshwater mussel (p<0.05 for each cases). Negative correlations were observed between the lipid peroxidation and glutathione or catalase levels after deltamethrin exposure, indicating a protective role of glutathione and catalase against lipid peroxidation, suggesting the use of these antioxidants as a potential biomarker of toxicity associated with contaminant exposure in freshwater mussels.

Glutamate cysteine ligase (GCL) in the freshwater bivalve Unio tumidus: impact of storage conditions and seasons on activity and identification of partial coding sequence of the catalytic subunit.

Glutamate cysteine ligase (GCL; EC 6.3.2.2) is the first enzyme involved in the synthesis of glutathione. A HPLC method with fluorimetric detection was used to measure GCL activity in the gills and the digestive gland of the freshwater bivalve, Unio tumidus. Storage conditions were optimized in order to prevent decrease of GCL activity and consisted in freezing the cytosolic fraction in the presence of protease (1 mM phenylmethylsulfonic fluoric acid) and gamma-glutamyltranspeptidase (1 mM L-serine borate mixture and 0.5 mM acivicin) inhibitors. Seasonal variations of activity in the digestive gland and to a lesser extent in the gills were found with activity increasing in spring compared to winter. No sex differences were revealed. The GCL coding sequence was identified using degenerated primers designed in the highly conserved regions of the catalytic subunit of GCL. The partial sequence identified encoded for 121 amino acids. The comparison of the identified partial coding sequence of U. tumidus with those available from vertebrates and invertebrates indicated that GCL sequence was highly conserved.

Antioxidative and oxidative changes in the digestive gland cells of freshwater mussels Unio tumidus caused by selected phenolic compounds in the presence of H(2)O(2) or Cu(2+) ions.

Research on biomarkers as early bioindicators of perturbation in populations and individuals has received increasing interest during recent decades. These ecotoxicity studies allow us to measure the impact of environmental stressors and to monitor and evaluate the degradation or restoration of systems. In the present study we used bivalve molluscs (mussels), which are sensitive biomarkers of aquatic ecosystem pollution, to assess the effects of three polyphenols: tannic acid, ellagic acid and gallic acid. These compounds were used in the 1-60 microM concentration range, alone and in the presence of H(2)O(2) (40 and 100 microM) or Cu(2+) ions (50 microM). The fluorescence probe dichlorofluorescein-diacetate (DCFH-DA) was used to measure reactive oxygen species (ROS). The oxidation of DCFH-DA to the fluorescent DCF (dichlorofluorescein) by the phenolic compounds was investigated spectrofluorimetrically. The results showed that the polyphenols tested can act as antioxidants against the ROS present in the digestive gland cells, but their activity is decreased after incubation with hydrogen peroxide or copper ions. SH-groups were determined spectrophotometrically using Ellman's reagent. The results showed that oxidative modification of proteins increased in a concentration-dependent manner in cells incubated with polyphenols (above 15 microM) alone. Incubation of the cells with phenolic acids and H(2)O(2) or Cu(2+) ions revealed that the phenolic acids had prooxidant properties in all concentrations used except for 1 microM tannic and ellagic acid and 40 microM H(2)O(2). DNA fragmentation was estimated by a fluorescence method using Hoechst 33258/propidium iodine binding. The data showed that the phenolic acids alone and in the presence of hydrogen peroxide or copper ions can induce apoptosis and necrosis. The methods used and results obtained indicate that the polyphenols selected can act not only as antioxidants but also as prooxidants in digestive gland cells of Unio tumidus.