Pollution impact on metal and biomarker responses in intestinal cytosol of freshwater fish.

In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fish collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue responsible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly influenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius vardarensis; Karaman, 1928), using for the first time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fish under the influence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fish from mining-affected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems.

The effect of different pollutants exposure on the pigment content of pigmented macrophage aggregates in the spleen of Vardar chub (Squalius vardarensis Karaman, 1928).

Pigmented macrophage aggregates (MAs) are known to change under influence of various factors, such as aging, season, starvation, and/or pollution. In this study, changes in the pigment content of the MAs in the spleen of Vardar chub (Squalius vardarensis, Karaman) (n = 129) collected in spring and autumn, from three rivers with different pollution impact was examined: Zletovska River (metals), Kriva River (metals and municipal wastewater), and Bregalnica River (municipal wastewater). Collected data revealed increased relative volume and number of MAs containing hemosiderin under the influence of metals, significant in autumn (p < .05). In chub exposed to metals combined with municipal wastewater, significant increase of lipochrome accumulation in MAs in autumn, melanin in MAs in fish captured in the spring season, and number of splenic MAs containing combination of melanin and lipochrome was noted. Volumes and number of MAs containing combination of hemosiderin and lipochrome increased in spleen of fish captured in autumn from both Zletovska River and Kriva River, most likely due to the contribution of hemosiderin and lipochrome, respectively. Values measured for the various pigments in splenic MAs in fish captured from Bregalnica River, were overall closer to the values measured for fish captured from Kriva River. Notably, melanin and lipochrome are more likely to be found in fish from waters influenced by municipal wastewater (organic pollution) and hemosiderin in fish spleen from water influenced by mining activity (heavy metals pollution).

Antioxidant and anti-inflammatory effects of the monocarbonyl curcumin analogs B2BRBC and C66 in monocrotaline-induced right ventricular hypertrophy.

For 22 days after monocrotaline injection two groups of rats received either of the monocarbonyl curcumin analogs (2E,6E)-2,6-bis(2-bromobenzylidene)cycloxehanone (B2BrBC) and (2E,6E)-2,6-bis([2-trifluoromethyl]benzylidene)cyclohexanone (C66), and their right ventricle parameters were compared to those from the control and the monocrotaline injected animals. B2BrBC and C66 treatments did not prevent the monocrotaline-induced right ventricular hypertrophy but attenuated the changes in antioxidant enzyme activities and reduced inflammation. The level of thiol-based nonenzymatic antioxidants did not change in the function of monocrotaline or curcumin analogs treatment. However, due to its stronger antioxidant properties, only B2BrBC treatment was effective in the reduction of monocrotaline-associated lipid peroxidation. The obtained results suggest that increasing the levels of antioxidant enzymes may not be sufficient to reduce oxidative stress and chronic inflammation optimally and our current study supports the potential of compounds with more than one beneficial biological activity as a promising treatment against the progression of cardiac hypertrophy.

Characterization and identification of selected metal-binding biomolecules from hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928) using various techniques of liquid chromatography and mass spectrometry.

Metals play crucial physiological roles, but they can also cause irreparable toxic effects through binding to important cellular biomolecules in aquatic organisms. The aim of this study was to determine the exact molecular masses and to identify several selected metal-binding biomolecules in hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928). Methods applied for the achievement of this goal were SEC-AEC-HPLC for two-dimensional separation of cytosolic biomolecules, HR ICP-MS for metal measurements, and mass spectrometry (MALDI-TOF-MS and LC-MS/MS) for biomolecule mass determination and identification. The analyzed biomolecules included: Fe-binding biomolecules, which were identified as hemoglobin subunit ß in the liver (molecular masses of ∼15 kDa), and hemoglobin subunits α and ß in the gills (molecular masses of ∼11 kDa, ∼13 kDa and ∼15 kDa); heat-stable Cd-binding biomolecules, which were identified as MT isoforms MT-I and MT-II (molecular mass of ∼6.0 kDa in both liver and gills, and an additional 4.9 kDa isoform in the gills); and heat-stable Mo-binding biomolecules of molecular masses equal to 3.3 kDa (in the gills) and 8.5 kDa (in the liver). An important finding of this study was the obvious presence of different isoforms of the same biomolecules in the liver and gills. This was, among others, manifested through the absence of Zn binding to MTs only in the gills, indicating that the same type of biomolecule can be responsible for different functions in different organs. Thus, for better understanding of metal behaviour in aquatic organisms, it is crucial to identify cellular metal-binding biomolecules and their functions.

Mining waste as a cause of increased bioaccumulation of highly toxic metals in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928).

Freshwater contamination with mining waste can result with high concentrations of toxic metals in the water and in fish organs. In North-Eastern Macedonia, several rivers (e.g., Zletovska, Kriva) are exposed to acid mine drainage from active Pb/Zn mines. Previous studies confirmed high concentrations of dissolved metals in their water. This study was performed in liver and gills of Vardar chub (Squalius vardarensis Karaman, 1928) from three Macedonian rivers (Bregalnica, Kriva and Zletovska) in spring and autumn 2012. The aim was to establish if increased exposure to certain metals have resulted with their increased bioaccumulation. The concentrations of 19 elements were measured in cytosolic tissue fractions, to obtain information on metabolically available metal species. The following ranges of cytosolic concentrations of highly toxic elements were measured in the Vardar chub liver (in µg/L): Cd, 1.18-184; Cs, 0.25-25.4; Tl, 0.02-5.80; Pb, 0.70-61.1. Their ranges measured in the gills (in µg/L) were the following: Cd, 0.24-59.2; Cs, 0.39-24.4; Tl, 0.01-1.00; Pb, 0.65-87.2. Although the water of the mining impacted Zletovska River was highly contaminated with several essential metals, especially with Mn and Zn, the majority of essential elements (Na, K, Ca, Mg, Co, Cu, Fe, Mn, Mo, and Zn) did not reflect the exposure level. In contrast, seven nonessential elements reflected the level of exposure in the water. Significantly increased hepatic and gill concentrations of Cs, Rb, Sr, and Tl were detected in Vardar chub from the Zletovska River compared to the other two rivers, of Cd and Pb in the Zletovska and Kriva River compared to Bregalnica, and of V in the Bregalnica River compared to Zletovska and Kriva rivers. Observed significant metal bioaccumulation, in particular of highly toxic elements, as a consequence of exposure to water contaminated with mining waste points to necessity of intensified supervision of mining impacted rivers.

Accumulation of Heavy Metals in Some Organs in Barbel and Chub from Crn Drim River in the Republic of Macedonia.

Heavy metal pollution of aquatic ecosystems is worldwide problem with consequences on the aquatic organisms. The concentrations of selected heavy metals (Fe, Mn, Zn, Cu, Cd and Pb) were determined in liver, kidney, gills, muscle and bone of barbel (Barbus rebeli) and chub (Squalius squalius) from the river Crn Drim in Macedonia. In general, liver and kidney in both fish species contained the highest concentration of the investigated metals, while muscle showed lowest bioaccumulation of heavy metals. Analyses supported significant seasonal differences in some organs for some metals: highest value in spring-summer compared to autumn-winter period. Generally, metal concentration was higher in the barbel than chub. The concentration of metals in muscle are bellow permissible value and do not constitute any threat for human health due to the consumption of metal loaded fish.

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.

Malondialdehyde concentrations in the intestine and gills of Vardar chub (Squalius vardarensis Karaman) as indicator of lipid peroxidation.

A lipid peroxidation product, malondialdehyde (MDA), was studied in Vardar chub (Squalius vardarensis Karaman) as an indicator of oxidative stress, using native fish from three rivers in northern Macedonia: the mining-impacted Zletovska and Kriva rivers and the agriculturally impacted Bregalnica River. MDA concentrations were measured in the intestine in the spring and autumn of 2012 and in the gills in autumn. The aims of the study were to establish the type of contamination which provokes a more pronounced MDA increase, as well as the organ which more reliably reflects the occurrence of oxidative stress. MDA levels in the intestine in spring amounted to 3.29-155.8 nmol g-1 and in autumn to 4.85-111.1 nmol g-1, whereas MDA concentrations in the gills in autumn were 7.69-147.5 nmol g-1. Stronger influence of organic contamination on development of oxidative stress was observed in both organs, as seen from higher median MDA concentrations in autumn in fish from the highly pesticide-contaminated Bregalnica River (gills 78.4 nmol g-1; intestine 23.5 nmol g-1) compared to the highly metal-contaminated Zletovska River (gills 15.9 nmol g-1; intestine 17.4 nmol g-1). The response of the gills to contamination was twice stronger than that of the intestine. The majority of fish from the pesticide-polluted river had increased MDA in the gills, in contrast to only sporadically increased MDA in the intestine. Our results indicated that development of oxidative stress strongly depends on the selected fish organ and that the gills seem to be a better choice for monitoring oxidative stress than the intestine, due to their continuous and direct exposure to polluted river water.

Effects of heavy metal pollution on pigmented macrophages in kidney of Vardar chub (Squalius vardarensis Karaman).

Pollution with heavy metals may influence the immune system of fish, leading to impairment of their health or even increase their mortality. The fish kidney is one of the first fish organs to be affected by water contamination. Amounts of kidney macrophages (MACs), which are involved in fish immune response, as well as the qualitative and quantitative changes in the pigmented MACs in fish kidney, are used as biomarkers of pollution. Therefore, in this study, we have evaluated relative and total volumes of trunk kidney pigmented MACs, and analyzed the pigments accumulated within them. Fish were sampled from two mining impacted rivers, Kriva and Zletovska, highly contaminated with heavy metals, and from one reference river, Bregalnica, in spring and autumn of 2012. We have observed that main pigments found in kidney MACs of Vardar chub were melanin and lipofuscin/ceroid, as well as that relative volumes of MACs ranged from 0.56 to 1.68%. Moreover, the results showed that relative volumes of pigmented MACs were higher in metal contaminated rivers, especially in autumn season in the Zletovska River, concurrently with extremely high metal exposure. In addition, condition factors and kidney somatic indices were found significantly lower in the Zletovska River in both seasons, autumn and spring, possibly also as a consequence of high water pollution. Our data confirm that increase in relative volumes of pigmented MACs may serve as warning sign of potential heavy metal pollution in aquatic environment.

Frequencies of erythrocyte nuclear abnormalities and of leucocytes in the fish Barbus peloponnesius correlate with a pollution gradient in the River Bregalnica (Macedonia).

Integrated chemical and biomarker approaches were performed to estimate if there is ongoing toxicity in the River Bregalnica, namely connected with the presence of metals. The study was performed in water, sediment, and barbel (Barbus peloponnesius), collected in two seasons, from two suspected polluted and one reference zones. The water analyses revealed higher mean values in polluted sites for most of the examined physicochemical parameters. Metal concentrations (Zn, Cu, Cd, Mn, Pb, and Fe) in water were more or less constant, whereas in sediment, they were higher at the two polluted locations. Condition factor (CF), as a general health indicator, revealed better overall condition in barbel from the reference site. In general, blood parameters revealed higher values in the polluted localities. Irrespective of sex and/or season, the frequency of micronuclei (MN) and vacuolated nuclei (VN) were with higher rates in polluted sites. Similarly, the frequencies of the leucocytes (Le), binuclei (BN), and irregularly shaped nuclei (ISN) were also significantly increased in the polluted localities, but they seemed prone to be influenced by sex and/or season. However, strong positive correlations between blood biomarkers and most water physicochemical parameters and metal in sediment were estimated. Our data support that the River Bregalnica's lower course receives significant genotoxic pollution, likely via metal industry effluents, agricultural runoff, and domestic sewage, and reinforced the utility of MN and other nuclear abnormalities as sensitive and suitable biomarkers for genotoxicity when used in monitoring studies.

Broad spectrum screening of 463 organic contaminants in rivers in Macedonia.

Target screening of 463 organic contaminants in surface water using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) with direct injection was performed in spring of 2015 in northern Macedonia, at six sampling sites in four rivers belonging to Vardar basin: Kriva, Zletovska, Bregalnica and Vardar. The aim of the study was to differentiate between various types of organic contamination characteristic for different types of anthropogenic activities, such as mining, agriculture, and urbanization. Depending on the studied river, 9-16% of analyzed compounds were detected. The highest total levels of organic contaminants were recorded in agriculturally impacted Bregalnica River (1839-1962ngL-1) and Vardar River downstream from the city of Skopje (1945ngL-1), whereas the lowest level was found in the mining impacted Zletovska River (989ngL-1). The principal organic contaminants of the Bregalnica River were herbicides (45-55% of all detected compounds; 838-1094ngL-1), with the highest concentrations of bentazone (407-530ngL-1) and molinate (84-549ngL-1), common herbicides in rice cultivation. The main organic contaminants in the other rivers were drugs (70-80% of all detected compounds), with antibiotics as a predominant drug class. The highest drug concentrations were measured in the Vardar River, downstream from Skopje (1544ngL-1). Screening of surface water by UHPLC-QTOF-MS was proven as a practical tool for fast collection of comprehensive preliminary information on organic contamination of natural waters, which can present a significant contribution in the monitoring and preservation of good ecological status of freshwater ecosystems.

Seasonal changes in hepatocytic lipid droplets, glycogen deposits, and rough endoplasmic reticulum along the natural breeding cycle of female ohrid trout (Salmo letnica Kar.)-A semiquantitative ultrastructural study.

This study on wild female Ohrid trout was primarily designed to provide a general overview of the breeding cycle influence upon selected aspects of hepatocytes. According with a semiquantitatively evaluation, some of these cell's structural compartments change during the breeding cycle. Structural modifications were disclosed in the relative occurrence of lipid, glycogen, and RER content during breeding cycle. The relative amount of lipid deposits in the hepatocytes was much greater in previtellogenesis, and decreased postspawning. So, while the seasonal changes in RER were positively related with the ovary maturation status, those of the lipid droplets followed an opposite trend. The hepatocytic glycogen occurred rarely, mainly in late-vitellogenesis and spawning, suggesting that in this species such kind of energy storage is comparatively unimportant. Lipid accumulation and later usage is, probably, the relevant biochemical pathway for Ohrid trout in the wild. While glycogen and RER contents were positively correlated with the gonadosomatic index, lipids were negatively correlated. Additionally, glycogen inclusions were positively correlated with the plasma estradiol levels. When comparing seasonal patterns from wild Ohrid trout with those from well-studied rainbow and brown trout (specimens studied were from aquaculture), there are contradicting results as to lipid and glycogen reserves, and also as to RER loads. The differences among the mentioned trout can result from intrinsic interspecies differences or may be associated with natural feeding conditions versus feeding with commercially prepared diets, or other factors. This study offers new data useful as standard to access liver pathology in wild and aquacultured Ohrid trout. Microsc. Res. Tech. 79:700-706, 2016. © 2016 Wiley Periodicals, Inc.

Histopathology investigation on the Vardar chub (Squalius vardarensis) populations captured from the rivers impacted by mining activities.

Many natural freshwater ecosystems, especially in the north eastern Macedonia, are polluted with heavy metals, which are released by active mines. Long-term exposure to high levels of dissolved metals might result in increased metal bioaccumulation in organs of aquatic organisms, and consequently might cause various sub-toxic and toxic effects. The aim of this study was to assess the health of Vardar chub (Squalius vardarensis) inhabiting mining impacted rivers Zletovska and Kriva, in comparison with chub from the reference Bregalnica River. It was done by use of indicators of tissue damage (histopathology of liver and gonads) and general indicators of exposure to environmental stressors (condition factor, organo-somatic indices and external/internal macroscopic lesions). Histological assessment of gonads revealed good reproductive health in all three rivers, indicating high tolerance of gonads to contaminant exposure. Contrary, several external/internal lesions were more pronounced in chub from severely metal contaminated Zletovska River. Prevalence of hepatic lesions was also higher in mining impacted rivers (in Kriva, 70%; in Zletovska, 59%) compared to Bregalnica River (38%). The spectrum of histological lesions observed in chub liver varied from non-specific minor degenerative conditions, such as lymphocyte infiltration, fibrosis, parasites, granulomas and lipidosis, to extensive and/or more severe changes such as bile duct proliferation, necrosis, megalocytosis, light-dark hepatocytes and hepatocytes regeneration. The results of histopathological investigation for all three rivers showed clear signs of water contamination, especially prominent in mining influenced rivers. More research efforts should be devoted to study of environmental conditions and metal contamination in the mining impacted rivers worldwide, especially of their effects on health of local ichthyofauna.

Evaluation of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) as an indicator of river pollution.

Quantification of histopathological alterations in the gills of Vardar chub (Squalius vardarensis Karaman) was performed in 2012 in rivers of north-eastern Macedonia, with the aim to examine the effects of water quality in the rivers (Zletovska and Kriva River-impacted by active Pb/Zn mines; Bregalnica River-contaminated by agricultural waste). The biological alterations in chub were classified as: circulatory disturbances, regressive and progressive changes, but their severity differed. Altogether the mildest changes were observed in the gills of chub from the Bregalnica River, a less polluted river, whereas mining impacted rivers were characterized by more severe alterations. In the gills of chub from the Zletovska River, which is highly contaminated with numerous metals, sulphates and chlorides, the highest lesion indices were found for the regressive changes of both epithelium and supporting tissue, with typical lesions referring to atrophy, thinning and lifting of epithelial cells, necrosis of epithelium and chloride cells, as well as deformations of lamellar cartilaginous base. Gill damages of chub from the Kriva River were overall milder compared to the Zletovska River, in accordance with pollution status. In the gills of chub from that river, progressive changes were more pronounced, specifically severe hyperplasia of mucous cells and epithelium in the interlammellar space, leading to fusion of lamellae, as well as hypertrophy of chloride cells. The comparison between seasons indicated higher intensity of progressive changes in all three rivers in autumn, when water level was very low, and consequently, water contamination was more pronounced due to concentration effect. The pattern and severity of histopathological alterations in the chub gills reflected differences in contamination levels and type of contaminants in different rivers and sampling periods, and thus have been proven as a valuable indicator of water quality.

Changes in the amount of kidney pigmented macrophage aggregates throughout the breeding cycle of female Ohrid trout, Salmo letnica Kar. (Teleostei, Salmonidae).

Changes in fish macrophages (Macs) are useful indicators of environmental pressures, but responses due to chemical and nonchemical stresses may be confounded by natural sources of variability. These may include sex and gonadal stage. In this study, we start addressing the following question: is the seasonally dependent ovary stage a factor to consider when using kidney Macs as biomarkers? To tackle this problem, the relative amount of pigmented Macs in kidney (head, trunk, and tail portions) was stereologically estimated in Ohrid trout, and related with the breeding status. The amount of Macs significantly increased from pre vitellogenesis to late vitellogenesis and showed a decreasing trend then after, with lower values noted after spawning in the head (1.9% versus 7.5% versus 2.0%), trunk (1.8% versus 7.5% versus 2.5%), and tail (2.5% versus 6.7% versus 2.9%) kidney. The decrease seen at spawning was significant in head and trunk kidney, and at post spawning it was significant for all kidney portions. The amounts of Macs were positively correlated with the ovary relative weights and plasma estradiol levels. We proved for the first time that fish kidney pigmented Macs can vary in amount during the breeding cycle. Our data, combined with literature, strongly support that the sex-steroid profile and kidney status-seasonal remodeling both influence the Macs pool; likely not only in female trout. So, while increases in Macs may warn of ecosystem problems, we show that using kidney Macs for biomonitoring should also take into account seasonally, particularly that related with ovary maturation.