Iron-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius): two-dimensional chromatographic separation with mass spectrometry detection.

Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-ß subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.

Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper.

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.

The study of acidic/basic nature of metallothioneins and other metal-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius).

Since fish metalloproteins are still not thoroughly characterized, the aim of this study was to investigate the acidic/basic nature of biomolecules involved in the sequestration of twelve selected metals in the soluble hepatic fraction of an important aquatic bioindicator organism, namely the fish species northern pike (Esox lucius). For this purpose, the hyphenated system HPLC-ICP-MS was applied, with chromatographic separation based on anion/cation-exchange principle at physiological pH (7.4). The results indicated predominant acidic nature of metal-binding peptides/proteins in the studied hepatic fraction. More than 90 % of Ag, Cd, Co, Cu, Fe, Mo, and Pb were eluted with negatively charged biomolecules, and >70 % of Bi, Mn, and Zn. Thallium was revealed to bind equally to negatively and positively charged biomolecules, and Cs predominantly to positively charged ones. The majority of acidic (negatively charged) metalloproteins/peptides were coeluted within the elution time range of applied standard proteins, having pIs clustered around 4-6. Furthermore, binding of several metals (Ag, Cd, Cu, Zn) to two MT-isoforms was assumed, with Cd and Zn preferentially bound to MT1 and Ag to MT2, and Cu evenly distributed between the two. The results presented here are the first of their kind for the important bioindicator species, the northern pike, as well as one of the rare comprehensive studies on the acidic/basic nature of metal-binding biomolecules in fish, which can contribute significantly to a better understanding of the behaviour and fate of metals in the fish organism, specifically in liver as main metabolic and detoxification organ.

Accumulation of metal(loid)s in the digestive gland of the mussel Unio crassus Philipsson, 1788: A reliable detection of historical freshwater contamination.

The possible influence of historical contamination of water/sediments on the metal(loid) bioaccumulation in the digestive gland of mussel Unio crassus Philipsson, 1788, from two differently contaminated sites at the Mreznica River was studied in three seasons. The first data for this species on total/cytosolic concentrations of 27 (non)essential elements were obtained by HR ICP-MS. Higher bioaccumulation was observed at the historically contaminated site, with several nonessential elements (Bi, Cs, Pb, Sb, Tl, U) found in 5-6 times higher concentrations compared to the reference site. Although both total and cytosolic levels revealed the influence of water/sediment contamination, the latter showed association between bioaccumulation and exposure for larger number of studied elements. At the reference site, several elements (Ba, Ca, Cd, Cr, Mn, Sr) were also found in 2-10 times higher concentrations compared to contaminated one, but it was attributed to background levels characteristic for karst rivers (for Ca and Cd), and to coaccumulation due to chemical similarity (for Ba, Cr, Mn, Sr). The seasonal variability was also observed, with generally highest metal(loid) concentrations in mussel digestive glands found in autumn which was associated to mussels reproductive period. Our results confirmed that sediment-dwelling mussels, specifically U. crassus, represent a good bioindicators for detection of historical pollution due to their direct contact/exposure to contaminants stored in sediments, with concurrent consideration of physiological/chemical factors. Historical contamination potentially can have serious impact on freshwater environment even long time after its cessation, and, therefore, a careful continuous monitoring is recommended.

Dynamics of drug contamination of the river-water in the rural, semirural and urban areas of the Mreznica River in Croatia during COVID-19 pandemic (2020-2021).

Increased contamination of aquatic ecosystems with pharmaceuticals could have been expected due to the COVID-19 pandemic. Surface water from three domains (rural, semirural, urban) of the Mreznica River (Croatia) was screened for 253 pharmaceuticals by SPE-UHPLC-MS/MS. At the beginning of the pandemic, the highest concentration of drugs (excluding veterinary) was detected at urban site (291.4 ng/L), followed by semirural (186.5 ng/L) and rural (141.6 ng/L). With the progression of pandemic, contamination increase was observed at all sites, but it was the most obvious at semirural (approximately 400-700 ng/L). The most pronounced concentration increases were observed for non-opioid analgesics, especially ibuprofen. In September 2021, the first notable occurrence of opioid analgesics was recorded. The most represented group of pharmaceuticals at the start of the pandemic (May 2020) was generally stimulants (caffeine, cotinine). In September 2021, the predominant group was analgesics at all sites (45-84%), whereas stimulants decreased to undetectable levels. The results of this study indicated that the epidemiological measures and medical treatments that were widely imposed/applied caused notable increase of the surface water contamination with drugs of a small river with limited dilution capacity, indirectly pointing to the changes that occurred in the behaviour and habits of the inhabitants of the affected areas.

Metal-binding biomolecules in the liver of northern pike (Esox lucius Linnaeus, 1758): The first data for the family Esocidae.

Metal-handling strategies of various fish species are known to vary significantly in association with their intracellular metal behaviour. Thus, to better understand the possible consequences of increased metal exposure in fish it is important to perform comparative studies on metal-binding biomolecules in organs of different species. This study was the first of this kind on a liver of an esocid fish (northern pike, Esox lucius), and the gathered information were compared to fish belonging to three other families, Leuciscidae, Cyprinidae and Salmonidae. Distributions of ten elements among cytosolic biomolecules of different molecular masses were studied by size exclusion HPLC combined offline with high resolution ICP-MS. The results indicated predominant association of Co, Fe and Mo to high molecular mass biomolecules (>100 kDa), of Zn and Bi to both high and medium molecular mass biomolecules (>30 kDa), of Mn and Se to medium molecular mass biomolecules (30-100 kDa), and Ag, Cd and Cu to low molecular mass biomolecules (10-30 kDa), presumably metallothioneins. Evident binding to metallothioneins was also detected for Zn and Bi. For several metals, distinct differences were observed when cytosolic metal distributions of northern pike were compared to leuciscids, salmonids and cyprinids. More pronounced Zn binding to metallothioneins was recorded in leuciscids and cyprinids than both esocids and salmonids, whereas cytosolic Mn and Se distributions clearly differed between all studied fish families. Accordingly, in assessment of metal pollution it is vital to consider the exposed species, which requires prior comprehensive comparative research on numerous aquatic organisms.

Yesterday's contamination-A problem of today? The case study of discontinued historical contamination of the Mreznica River (Croatia).

The remnants of historical industrial contamination can be detected in many aquatic ecosystems worldwide even at present time. Mreznica is a river in Croatia that has been, for more than a hundred years, continually exposed to effluents of various industries, which have, in modern time, mostly ceased to operate. Our aim was to establish the level of current contamination and pollution of the Mreznica river-water and sediments. The study of river contamination at three sites (reference site; site nearby former cotton industry facility in Duga Resa - DRF; industrial zone of Karlovac town - KIZ) in three sampling campaigns (May 2020, April and September 2021) encompassed analyses of physico-chemical water parameters, screening of 369 pesticides, measurement of metal (loid) concentrations in the sediments, and in the dissolved and particulate phases of the river-water. The sediment pollution was assessed through the analyses of total bacteria abundance (by targeting 16S rRNA genes), and their associated metal resistance genes (cnrA, pbrT and czcD) and class 1 integrons (intl1). At the DRF site, industrial organic contaminants that can be traced to textile production were detected (dye and nylon components), as well as increased levels of some metals bound to suspended particulate matter and sediments. At the most downstream KIZ site, occasional high level of industrial herbicide neburon was measured in the river-water, and metal contamination of suspended particulate matter and sediments was evident. Although, based on the comparison with legislation and literature data, the level of contamination was rather mild, the effects on microbial communities were unquestionable, confirmed by increased abundance of the czcD gene at DRF site and the intI1 gene at both industrially impacted sites. Obtained results indicated long-term sediment retention of some industrial contaminants at the places of historical freshwater contamination, and, thus, the necessity for their monitoring even after the termination of contamination sources.