Heart rate variation and DNA damage in haemocytes of the mediterranean mussel Mytilus galloprovincialis L. as biomarkers of marine pollution

Integrisani pristup biomonitoringa koji uključuje bateriju biotestova, omogućava pouzdaniju detekciju različith vrsta organskog i neorganskog zagađenja u cilju procjene stanja u morskom ekosistemu. U ovom radu je praćen uticaj hemijskih stresora na srčani ritam mediteranske dagnje pri kratkotrajnim tretmanima ex situ. Pošto biomarkeri na ćelijskom i molekularnom nivou imaju veću specifičnost, u cilju unaprijeđenja pouzdanosti i praktične primjenljivosti srčanog ritma kao fiziološkog biomarkera, uradjeni su i produženi tretmani dagnji, takođe ex situ, u kojima su uporedno analizirani srčani ritam i oštećenja DNK u hemocitama mediteranske dagnje. Pri kratkotrajnim tretmanima, ispitivan je uticaj promjene u salinitetu morske vode na srčani ritam dagnji koji je praćen fotopletizmografski, fiber-optic metodom. Takođe, ispitivan je odgovor srčanog ritma dagnje pri izlaganju kadmijum hloridu, dizel gorivu, naftnom disperzantu Superdispersant-25 i dispergovanom dizelu, pri čemu je potvrđena sposobnost dagnji da detektuju stres u ranoj fazi. Prilikom produženih tretmana dagnji, uporedno sa praćenjem srčanog ritma, procijenjen je genotoksični potencijal kadmijum hlorida kao model mutagena, dizel goriva, Superdispersant-25, benzo(a)pirena i tributil kalaj hlorida kao zagađivača čija kontrola je od velikog značaja. Uz komet test koji je služio za procjenu oštećenja DNK, genotoksični potencijal tributil kalaj hlorida, analiziran je mikronukleus testom i prokariotskim SOS/umuC testu in vitro. Dobijeni rezultati su pokazali da je dispergovani dizel mnogo opasniji po školjke usljed veće dostupnosti njegovih sastojaka. Rezultati praćenja srčanog ritma pri tretmanima tributil kalaj hloridom mogu se povezati sa podacima dobijenim u genotoksikološkim testovima što ukazuje da bi se srčani ritam dagnji mogao koristiti kao pouzdan biomarker u detekciji zagađenja porijeklom od organokalajnih jedinjenja. Na osnovu rezultata dobijenih pri kratkotrajnim i produženim tretmanima organskim i neorganskim zagađivačima, srčani ritam mediteranske dagnje se može preporučiti za korišćenje kao sastavni dio integrisanog pristupa u biomonitoringu zagađenja mora.reliable detection of different types of organic and inorganic pollutants which would improve the evaluation of the state of the marine ecosystem. In this study, the influence of short-term treatments by chemical stressors on the heart rate of the Mediterranean mussel was monitored ex situ. Since cellular and molecular biomarkers are characterized by higher specificity, extended treatments for comparative analyses of the heart rate and DNA damage in haemocytes were performed to improve reliability and applicability of the Mediterranean mussel’s heart rate as a physiological biomarker. Fiber-optic method, based on photoplethysmography, was applied for studying the influence of variations in seawater salinity on the heart rate of mussels. The influence of cadmium chloride, diesel oil, oil dispersant Superdispersant-25 and dispersed diesel oil was studied as well, whereby the ability of mussel’s heart rate to detect stress at an early stage was confirmed. Comparative analyses of mussel’s heart rate and genotoxic potential of cadmium chloride as a model mutagen, diesel oil, Superdispersant-25, benzo(a)pyrene and tributyltin as pollutants of importance to control, in extended treatments were assessed. Comet assay was used for the assessment of DNA damage. Moreover, genotoxic potential of tributyltin was assessed in vitro by micronucleus test and prokaryotic SOS/umuC assay. The results indicated that dispersed diesel oil was more harmful for mussels due to enhanced availability of its compounds. Heart rate results obtained in tributyltin chloride treatments can be linked to the data obtained in genotoxicological assays indicating that musels‘s heart rate could be used as a reliable biomarker for detection of organotin pollution. The results obtained in short-term and extended treatments by organic and inorganic pollutants indicated that heart rate of the Mediterranean mussel can be included as a part of the integrated approach of marine pollution biomonitoring

Evaluation of Physiological State of Pen Shell Pinna nobilis (Linnaeus, 1758) by a Non-Invasive Heart Rate Recording under Short-Term Hyposalinity Test

A non-invasive laser fiber-optic method based on infrared sensors for heart rate (Hr) recording was applied to assess the physiological condition of Pinna nobilis. During 2017, the specimens of P. nobilis were sampled at three sites within the Boka Kotorska Bay, Montenegro and used for ex situ experiments with short-term reduction/restoration of ambient salinity to evaluate their physiological adaptive capacity based on heart rate recovery time (Trec). Mean Trec for specimens from Sv. Nedjelja (reference site), Dobrota and Sv. Stasije were 72 ± 3, 91 ± 7 and 117 ± 15 min, while the coefficients of variation (CV) were 0.12, 0.13 and 0.17, respectively. Resting heart rate (Hrrest) and Trec showed statistically significant differences between the groups of mussels from Dobrota and Sv. Stasije in comparison to the reference site. Statistically significant correlations were observed between Trec and shell length/width, which was not the case in comparison between Hrrest and shell length/width. The lower adaptive capacity within the P. nobilis specimens from Dobrota and Sv. Stasije in comparison to the reference site could occur due to stress induced by deterioration of environmental conditions, which could have led to impairment of the physiological state of the mussels evaluated by Hr. All the specimens of P. nobilis survived the experimental treatments; afterwards, they were successfully transplanted at the Dobrota site. The experimental unit with sensor technology applied in this study can provide Hr recording in real time and could have an application in monitoring the physiological/health state of P. nobilis individuals maintained in aquaria

Transplant caging of seabream (Sparus aurata) as a monitoring tool for marine pollution assessment in the Montenegrin Adriatic coast

More frequent application of biological parameters beside the conventional chemical parameters in national monitoring for marine ecosystem assessment is supported by UNEP/MAP. Accordingly, to evaluate the possible effects of pollution by determination of trace element content and genotoxicity assessment in marine organisms within the Boka Kotorska Bay, Montenegro (southern Adriatic), we carried a field study which included transplantation of the seabream (Sparus aurata) from aquaculture farm (Orahovac) to more impacted sites (Dobrota and port of Tivat), situated in vicinity of the main ports. On the sampling site Orahovac, the group of seabream specimens were placed in closed fish trap on 5 m depth (O1), another fish trap was placed in Dobrota, while the third fish trap with seabreams was placed at the site port of Tivat. The additional group was sampled from standard aquaculture cage on sampling site (O2) to reduce possible bias in genotoxicity data due to confined space within the fish trap. After two weeks of exposure, blood from fish heart was taken, afterwards muscle tissues were prepared for trace element determination. Genotoxicity was measured by DNA damage induction based on comet (single cell gel electrophoresis) assay parameter – Tail intensity (TI%), while trace element (As, Cd, Pb, Hg, Cr, Cu, Fe, Mn, Ni, Zn and V) content was determined by ICP. Mean values of TI% obtained from seabream specimens at the sampling site Orahovac were: 4.26 ± 0.17 – O1 and 2.56 ± 0.11 – O2, while TI% values for transplanted specimens on the sites Dobrota and port of Tivat were 6.38 ± 0.17 and 11.06 ± 0.23, respectively. Statistically significant differences (p<0,05) of TI% were observed between all specimens groups. The group of seabream from port of Tivat showed the highest TI% (higher level of DNA damage), most probably caused by marine pollution. Obtained trace element concentrations in seabream tissues were significantly lower in comparison to values recommended by FAO, EC and national legislatives of Croatia, Spain and Turkey. Since transplanted fish showed a significant response in a relatively short exposure period, our results support the introduction of seabream caging as monitoring tool to reveal marine pollution by multiple biomarker approach. Considering the significant share of aquacultured seabream in human consumption, higher availability for sampling and convenience in application for molecular biomarker analyses in the same samples, such an integrated monitoring approach would contribute to more credible data

Express method for isolation of ready-to-use 3D chitin scaffolds from aplysina archeri (aplysineidae: verongiida) demosponge

Sponges are a valuable source of natural compounds and biomaterials for many biotechnological applications. Marine sponges belonging to the order Verongiida are known to contain both chitin and biologically active bromotyrosines. Aplysina archeri (Aplysineidae: Verongiida) is well known to contain bromotyrosines with relevant bioactivity against human and animal diseases. The aim of this study was to develop an express method for the production of naturally prefabricated 3D chitin and bromotyrosine-containing extracts simultaneously. This new method is based on microwave irradiation (MWI) together with stepwise treatment using 1% sodium hydroxide, 20% acetic acid, and 30% hydrogen peroxide. This approach, which takes up to 1 h, made it possible to isolate chitin from the tube-like skeleton of A. archeri and to demonstrate the presence of this biopolymer in this sponge for the first time. Additionally, this procedure does not deacetylate chitin to chitosan and enables the recovery of ready-to-use 3D chitin scaffolds without destruction of the unique tube-like fibrous interconnected structure of the isolated biomaterial. Furthermore, these mechanically stressed fibers still have the capacity for saturation with water, methylene blue dye, crude oil, and blood, which is necessary for the application of such renewable 3D chitinous centimeter-sized scaffolds in diverse technological and biomedical fields. © 2019 by the authors

Atypical hodgkin and reed-sternberg cells in peripheral blood of a patient with advanced stage of Hodgkin's disease - a case report

The occurrence of abnormal Hodgkin's and Reed-Sternberg cells in the peripheral blood in a patient suffering from Hodgkin's disease has been noticed exceptionally rare in a previous period, and especially rare in last ten years primarily due to successfull treatment of this disease. The presence of atypical mononuclear cells in peripheral blood which cytomorphologically resembled Reed-Sternberg cells was registered in 8 patients till 1966. During the last decade, the presence of atypical mononuclear cells in the peripheral blood was used for their isolation cultivation, and detailed immunophenotypic and genetic analysis. The analysis of mononuclear cells in rare patients with Hodgkin's disease was established that they belong to the B-lymphoid cells with expression of CD30 and CD15 antigens. The examination of presence of Hodgkin's cells in the peripheral blood of patients with Hodgkin's disease is important for patients with advanced stage of the disease in which autologous stem cell transplantation and high dose chmeotherapy is planned. The authors present a 33-year-old patient, who noticed enlarged neck lymph nodes in September 2000, high temperature and loss in weight. On physical examination enlarged neck lymph nodes 5x8 cm and hepatosplenomegaly were found. There was anemia and thrombo-cytopenia, and normal WBC count with 24% of lymphoid elements in differential formula. On histologic examination of lymph nodes Hodgkin¢s disease, type nodular sclerosis with mixed cellularity was found. Histology of bone marrow showed nodal lymphomatous infiltration. Immunohistochemistry with monoclonal antibodies of concentrate of peripheral blood cells showed expression of CD30+ and CD15+, immunophenotypically and morphologically matching Reed-Sternberg cells. Cytogentic analysis of mononuclear cells of the bone marrow showed normal karyotype. The patient was in clinical stage IV/V of the disease and chemotherapy with 9 cycles of ABVD+Mp protocol was applied. He is still in remission

Single and combined potential of polystyrene microparticles and fluoranthene in the induction of DNA damage in haemocytes of Mediterranean mussel (Mytilus galloprovincialis)

In this study, the possible ‘vector effect’ within the exposure of Mediterranean mussels (Mytilus galloprovincialis) to polystyrene microplastics with adsorbed fluoranthene was investigated by applying the multibiomarker approach. The major focus was placed on genotoxicological endpoints as to our knowledge there are no literature data on the genotoxicity of polystyrene microparticles alone or with adsorbed fluoranthene in the selected experimental organisms. DNA damage was assessed in haemocytes by comet assay and micronucleus test. For the assess- ment of neurotoxicity, acetylcholinesterase activity was measured in gills. Glutathione S-transferase was assessed in gills and hepatopancreas since these enzymes are induced for biotransformation and excretion of lipophilic compounds such as hydrocarbons. Finally, differences in physiological response within the exposure to polystyrene particles, fluoranthene, or particles with adsorbed fluoranthene were assessed by the variation of heart rate patterns studied by the noninvasive laser fibre-optic method. The uniform response of individual biomarkers within the exposure groups was not recorded. There was no clear pattern in variation of acetylcholinesterase or glutathione S-transferase activity which could be attributed to the treatment. Exposure to polystyrene increased DNA damage which was detected by the comet assay but was not confirmed by micronucleus formation. Data of genotoxicity assays indicated differential responses among the groups exposed to fluoranthene alone and fluoranthene adsorbed to polystyrene. Change in the heart rate patterns within the studied groups supports the concept of the Trojan horse effect within the exposure to polystyrene particles with adsorbed fluoranthene

Comparative assessment of cardiac activity and DNA damage in haemocytes of the Mediterranean mussel Mytilus galloprovincialis in exposure to tributyltin chloride

This study gives an insight in sensitivity of heart rate (Hr) of Mytilus galloprovincialis as a physiological biomarker. Impact of tributyltin chloride (TBT-Cl) on Hr was studied in parallel with evaluation of mutagenic, genotoxic and cytotoxic potential of TBT-Cl (10,100 and 1000 mu g/L) within 96 h treatment in static conditions. Mutagenic potential was assessed by SOS/umuC assay while genotoxicity was assessed in haemocytes of M. galloprovincialis by using the comet assay and the micronucleus test. Benzo(a)pyrene (B(a)P) was used as a positive control. Hr variations detected in TBT-Cl treatments can be linked to data obtained in the genotoxicological assays indicating that Hr can be considered and used as a reliable physiological biomarker for detecting the presence of organotin compounds. However despite the observed genotoxic potential of B(a)P, a noteworthy Hr response was not observed which further questions the potential of Hr in the detection of different types of pollutants

First insight into the whole genome shotgun sequence of the endangered noble pen shell Pinna nobilis : a giant bivalve undergoing a mass mortality event

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Pegylated interferon for treatment of chronic hepatitis C in hemodialysis patients in Croatia

Hepatitis C virus (HCV) infection is a frequent complication among long-term dialysis patients. The aim of the present study was to evaluate the efficacy and side effects of pegylated interferon-α(2a) (PEG-IFN-α(2a)) treatment in hemodialysis patients. We retrospectively reviewed charts of 16 HCV-RNA-positive hemodialysis patients. There were 11 male and 5 female patients treated with dialysis for 6-28 years. Twelve patients had HCV genotype 1b, 2 patients had 3a, and 1 patient had genotype 2a. Although only 10 out of 16 patients completed 48 weeks of treatment, early virological response and end-of-treatment virological response were achieved in 9 and 13 patients, respectively. Sustained virological response was recorded in 9 patients. The most common side effect was anemia. A flu-like syndrome was documented in 6, myalgia in 4, and arthralgia in 5 patients. Rectorrhagia, endocarditis and severe cough were recorded in 1 patient each. Nine patients received a renal transplant, and all 6 responders remained HCV-RNA-negative. PEG-IFN-α(2a) has limited efficacy in dialysis patients. A significant proportion of patients discontinued treatment because of side effects. Additional studies with long-term follow-up are needed to determine the optimal treatment of HCV infection in the dialysis population