Marijana Erk

Analysis of metallothioneins by the modified Brdička procedure

Quantitative analysis of metallothioneins (MTs) by the modified Brdicka procedure is based on the evaluation of the catalytic hydrogen signal (Cat). The effect of the basic physico-chemical parameters, the temperature and the depolarizer concentration (Co(NH(3))(6)Cl(3)), on the Cat signal height and the respective calibration straight lines was studied by means of the differential pulse (DP) mode at the hanging mercury drop electrode (HMDE). The temperature was varied from 7 to 18 degrees C, and the depolarizer concentration from 1.2x10(-4) to 1.0x10(-3) M Co(NH(3))(6)Cl(3). The commercially available rabbit liver MT was used as the calibrant. The parameters of the calibration straight lines, the standard errors of the intercept s(a) and the slope s(b) were evaluated by means of the least-square method. The hypothesis of a significant difference of the estimated value of the intercept on y-axis compared to the zero value was statistically tested. For those conditions for which this hypothesis was rejected the measuring conditions of the catalytic hydrogen signal are selected and they refer to a buffer 1M (NH(4)Cl+NH(4)OH), pH 9.5, (7.0+/-0.5) degrees C and the depolarizer concentration (0.8 or 1.0)x10(-3) M Co(NH(3))(6)Cl(3).

Cellular energy allocation in mussels (Mytilus galloprovincialis) from the stratified estuary as a physiological biomarker.

This study demonstrates the usefulness of cellular energy allocation (CEA) evaluations as a physiological biomarker to infer the occurrence of natural stress in native populations of mussels inhabiting the stratified estuary (Krka River estuary, Croatia). Sampling sites were selected based on their differences in the salinity and temperature. The CEA value was calculated as a ratio between available energy (E(a)) and energy consumption (E(c)). Generally, higher values of E(a) were recorded in June than in November, which were especially evident in the storage components (carbohydrates and lipids), while the constitutive component (proteins) remained relatively constant. The highest E(c) was recorded in mussels at estuarine site compared to coastal site, which may be caused by the energetically costly maintenance of osmotic balance. Decrease in CEA recorded in estuarine mussels may ultimately result in a lower amount of energy available for growth, reproduction, or defence against other stresses (e.g. pollution).

Protein and metal concentrations in two fractions of hepatic cytosol of the European chub (Squalius cephalus L.).

Indigenous specimens of European chub were caught at five sites in Sava River during two successive non-reproductive periods and used as bioindicator organisms for the assessment of metal accumulation. Liver was selected as a target organ and the metal concentrations (Zn, Cu, Fe, Mn, and Cd) were determined in two fractions of hepatic cytosol, namely in untreated (S50) and heat-treated (HT S50) fraction. In addition, the concentrations of total cytosolic proteins were measured in untreated (S50) fractions, whereas the concentrations of specific metal-binding proteins, i.e. metallothioneins, were determined in the heat-treated fractions. Concentrations of Zn and Fe were significantly higher in the untreated compared to heat-treated cytosol, because these metals are associated to the heat-sensitive metalloenzyme and high molecular mass proteins. On the contrary, Cu, Mn and Cd were predominantly present in the heat-treated cytosol, which contains metallothioneins. In both fractions of hepatic cytosol, nonessential metal Cd showed statistically significant increase from the upstream sites (median 5 ng mL(-1)) toward the downstream site Jasenovac (median 11 ng mL(-1)). The metallothionein concentrations, on the contrary, did not differ between sites, probably due to low dissolved and labile concentrations of metals known as metallothionein inducers (Zn, Cu, and especially Cd) in river water. Therefore, Cd concentrations in hepatic cytosol of European chub can be recommended as an early-warning marker of fish chronic exposure to Cd from combined sources, water and ingested food.

Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules.

Serum metallothionein in patients with testicular cancer.

AbstractPurpose Metallothioneins (MTs) have been disclosed as a useful diagnostic factor for tumour progression and drug resistance in a variety of malignancies. Increased levels of MT in blood serum have been found in patients with several types of cancer, but there is no available information on serum MT levels in patients with testicular germ cell tumour (TGCT). The aim of the study was to determine MT levels in serum of patients with TGCT and to evaluate the portion of platinum (Pt) that binds to MT after cisplatin administration since MTs could be involved in drug resistance.MethodsConcentration of total MT was determined in serum of 25 men with newly diagnosed TGCT by differential pulse voltammetry. The fractionation of serum was carried out by size exclusion high-performance liquid chromatography (SE-HPLC), while concentration of Pt in collected fractions was determined by inductively coupled plasma mass spectrometry.ResultsConcentration of serum MT was significantly higher in TGCT patients than in healthy volunteers. The results of SE-HPLC analysis showed that only a small amount of Pt was bound to proteins in the area of MT elution.ConclusionsSignificant increase in MT levels in individuals with TGCT indicates certain health problem and, in combination with other commonly used diagnostic tools, could improve early diagnosis.

The interactions of 54Mn with aminopolycarboxylic acids in aquatic systems

Abstract The interactions of 54 Mn radionuclide with ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) were studied in freshwater system (Butoniga impoundment), and in diluted seawater system (the Rasa Bay) by high voltage paper electrophoresis. Depending on the EDTA or NTA concentrations it was possible to detect the distribution of cationic, anionic and immobile 54 Mn species, and to calculate their electrophoretic mobilities. The apparent concentration stability constants of 54 Mn-EDTA or 54 Mn-NTA complexes were calculated from the respective titration curves. Logarithmic values of the apparent concentration stability constants for 54 Mn-EDTA complex corrected for the influence of pH, K c( α H) , are in the range 6.20–7.38 in two different water types, while corrected for the effect of pH and Ca 2+ , K c(αL) , in 5% seawater amount from 11.29 to 11.89. Logarithmic values of the apparent concentration stability constants for 54 Mn-NTA complex corrected for the influence of pH, K c( α H) , are in the range 6.42-5.21 in two different water types, while corrected for the effect of pH and Ca 2+ , K c( α L) , in 5% seawater amount from 6.92 to 6.47.

Assessment of metal exposure, ecological status and required water quality monitoring strategies in small- to medium-size temperate rivers

ABSTRACT The present study was undertaken to obtain a better understanding of the seasonal variability of total dissolved metal/metalloid levels and physicochemical parameters within small- to medium-size freshwater ecosystems in temperate climate region. The research was conducted in four seasons in the Sutla River, medium-size polluted, and the Črnomerec Stream, small-size unpolluted watercourse in Croatia. In the Sutla River, characterized by the rural/industrial catchment, physicochemical parameters and total dissolved metal concentrations of 21 trace and 4 macro elements were analysed downstream of the point source of pollution, the glass production facility, indicating for the first time their variability across four seasons. Based on dissolved oxygen, total dissolved solids, nutrient concentrations, conductivity and total chemical oxygen demand, quality status of the Sutla River was good, but moderate to poor during summer, what was additionally confirmed by the highest levels of the most of 25 measured metals/metalloids in summer. Comparison with the reference small-size watercourse, the Črnomerec Stream, indicated significant anthropogenic impact on the Sutla River, most evident for Fe, Mn, Mo, Ni, Pb, Rb and Tl levels (3-70-fold higher in the Sutla River across all seasons). Generally, presented results indicated significant decrease of the water quality in the anthropogenically impacted small- to medium-size watercourses in summer, regarding physicochemical water parameters and total dissolved metal/metalloid concentrations, and pointed to significant seasonality of these parameters. Confirmed seasonality of river ecological status indicates that seasonal assessment represents a prerequisite for proper classification of the water quality in small- to medium-size temperate rivers.

Investigation of the soluble metals in tissue as biological response pattern to environmental pollutants (Gammarus fossarum example).

In the present study, Gammarus fossarum was used to investigate the bioaccumulation and toxic effects of aquatic pollutants in the real environmental conditions. The novelty of the study is the evaluation of soluble tissue metal concentrations in gammarids as indicators in early assessment of metal exposure. In the Sutla River, industrially/rurally/agriculturally influenced catchment in North-Western Croatia, physico-chemical water properties pointed to disturbed ecological status, which was reflected on population scale as more than 50 times lower gammarid density compared to the reference location, Črnomerec Stream. Significantly higher levels of soluble toxic metals (Al, As, Cd, Pb, Sb, Sn, Sr) were observed in gammarids from the Sutla River compared to the reference site and reflected the data on higher total dissolved metal levels in the river water at that site. The soluble metal estimates were supplemented with the common multibiomarker approach, which showed significant biological responses for decreased acetylcholinesterase activity and increased total soluble protein concentrations, confirming stressed environmental conditions for biota in the Sutla River. Biomarker of metal exposure, metallothionein, was not induced and therefore, toxic effect of metals was not confirmed on molecular level. Comparable between-site pattern of soluble toxic metals in gammarids and total dissolved metal levels in water suggests that prior to biomarker response and observed toxic impact, soluble metals in tissue might be used as early warning signs of metal impact in the aquatic environment and improve the assessment of water quality.

Selection of Target Mussel Tissue for Application of Cellular Energy Allocation as a Physiological Biomarker in Native Mussels Mytilus galloprovincialis (Lamarck, 1819)

ABSTRACT Three selected mussel tissues (digestive gland, mantle, and gills) were studied to determine which was the most suitable for the potential use of the cellular energy allocation (CEA) methodology in indigenous mussels Mytilus galloprovincialis. In addition, the applicability of CEA in the assessment of natural stress caused by salinity fluctuations in stratified estuary was tested in selected tissues. It was important to identify the mussel gender to reliably assess the changes in organism energy budget. CEA value was calculated as a ratio between available energy (Ea) and energy consumption (Ec). Mantle tissue was under the strongest influence of the differences in protein and lipid content between male and female mussels, and therefore reflected physiological changes in the organism itself, rather than those caused by natural environmental stress. CEA in gills had lower values than in mantle and digestive gland, and was similar at two selected sampling sites, so the changes in CEA caused by natural stress could not be detected in the gill tissue. Greater Ec in mussels from the estuarine site than from the coastal site was detected only in the digestive gland tissue, and can probably be attributed to the energetically costly maintenance of osmotic balance. Last, using digestive gland tissue in CEA analysis demonstrated a clear difference between coastal and estuarine sampling sites, providing the measure of the natural stress posed by variations in salinity.

Size-exclusion HPLC analysis of trace element distributions in hepatic and gill cytosol of Vardar chub (Squalius vardarensis Karaman) from mining impacted rivers in north-eastern Macedonia

Many bioindicators have not yet been well characterized regarding their tendency to bind trace elements by different cytosolic biomolecules in response to trace element exposure. Accordingly, our principal aim was to define the cytosolic distributions of Cd, Co, Cu, Fe, Mn, Mo, Se, and Zn among the biomolecules of different molecular masses in liver and gills of Vardar chub (Squalius vardarensis Karaman), a representative fish species of Macedonian rivers, and to determine distribution changes which occur as a consequence of increased exposure to specific trace elements. Additionally, we aimed to confirm the presence of heat-stable biomolecules in chub hepatic and gill cytosols. Distribution profiles were obtained by separation of cytosols and heat-treated cytosols using size-exclusion high performance-liquid chromatography, and by offline determination of trace element concentrations using high resolution inductively coupled plasma-mass spectrometry. Distribution profiles of trace elements were mainly characterized by several peaks encompassing different ranges of molecular masses, as a sign of incorporation of trace elements in various biomolecules within hepatic and gill cytosols. Especially interesting finding was probable binding of Fe to ferritin, which was especially pronounced in the liver, as a sign of important liver function in Fe storage. Furthermore, association with heat-stable proteins, metallothioneins (MT), was indicated for Cd, Cu, and Zn in the hepatic cytosol, as well as for Cd in the gill cytosol, whereas a sign of Zn-MT association was not observed in the gills. The presence of Mo- and Se-binding heat-stable compounds of very low molecular masses (<10kDa) in the cytosol was determined for both liver and the gills. Trace elements under all studied conditions were found associated to the same biomolecules, and only their proportions associated to specific cytosolic compounds have changed as a consequence of their increased bioaccumulation in the liver and gills of Vardar chub.