Effects of white-tailed eagle (Haliaeetus albicilla) nestling diet on mercury exposure dynamics in Kopacki rit Nature Park, Croatia.

The present study assessed for the first time the magnitude and dietary ecological source of total mercury (THg) exposure in a southern population of white-tailed eagles (Haliaeetus albicilla), an apex predator species shown valuable for environmental biomonitoring. This population depends on the Kopacki rit Nature Park - the most important breeding site. We assessed THg exposure, using nestling body feathers collected between 2014-2019 (n = 72), and potential dietary ecological sources, proxied by prey remains and stable isotope analysis. Results show THg concentrations vary significantly over the years, though not showing any time trend. Prey remains analysis shows nests with aquatic prey remains to exhibit higher THg concentrations (median: 7.57 µg g-1 dw; min - max: 6.00-13.16 µg g-1 dw) compared to those with terrestrial remains (median: 3.94 µg g-1 dw; min - max: 0.28-12.04 µg g-1 dw) or evidencing a mixed diet (median: 7.43 µg g-1 dw; min - max: 3.38-12.04 µg g-1 dw). Nests with a predominant aquatic diet show elevated lower δ13C and higher δ15N values, indicating agreement between both dietary approaches. The model selection reveals a combination of year and δ15N best explain the variability in feather THg concentrations. Complementing these predictors with a dietary descriptor based on prey remains results in a poorer model fit and lowered explanatory power, similar to sexing the nestlings. The observed body feather THg concentrations (median: 6.99 µg g-1 dw; min - max: 0.27 - 17.16 µg g-1 dw) exceeded putative biogeochemical background levels (5.00 µg g-1 dw) in 71% of the nestlings, though, did not seem to exceed a threshold at which detrimental physiological effects are expected (40 µg g-1 dw). Continued monitoring is warranted as the studied population is likely exposed to a larger cocktail of contaminants while resident-protected bird areas.

Physiological Response of Nutrient-Stressed Lemna gibba to Pulse Colloidal Silver Treatment.

Wastewater is a source of many environmental pollutants and potentially high concentrations of essential plant nutrients. Site-specific nutrient levels may influence the response of exposed plants to a chemical stressor. In the present study, we focused on the responses of model aquatic macrophyte swollen duckweed (Lemna gibba L.) to a short pulse exposure and a commercially available colloidal silver product as a potential environmental chemical stressor, combined with two levels of total nitrogen and phosphorus nutrition. Treatment with the commercially available colloidal silver product caused oxidative stress in L. gibba plants under both high and low nutrient levels. Plants grown and treated under high nutrient levels showed lower levels of lipid peroxidation and hydrogen peroxide accumulation, as well as higher levels of photosynthetic pigment content in comparison to treated plants under low nutrient levels. Higher free radical scavenging activity for plants treated with silver in combination with high nutrient levels resulted in better overall protection from silver-induced oxidative stress. The results showed that external nutrient levels significantly affected the L. gibba plant's response to the colloidal silver presence in the environment and that nutrient levels should be considered in the assessment of potential environmental impact for contaminants.

ARSENIC IN DRINKING WATER AND URINE AND ITS RELATIONSHIP WITH MALIGNANT TUMORS OF URINARY TRACT IN OSIJEK-BARANJA COUNTY, CROATIA.

Increased values of arsenic in potable water in eastern Croatia has been a matter of scientific interest for the past two decades due to numerous health effects, including carcinogenic ones. This study investigated whether prolonged exposure to increased arsenic from water could be detectable through increased arsenic in urine, and whether it influenced the incidence of kidney and bladder cancer in Osijek-Baranja County. Inductively coupled plasma mass spectrometry (ICP-MS) was used for analysis of water samples from available water sources (wells, aqueducts). In addition, examinees from Osijek, Nasice, Vladislavci, Cepin and Dalj gave their urine samples for analysis. Data on cancer incidence were obtained from the Institute for Public Health Registry and cumulative incidence of kidney and bladder cancer was calculated for the period between January 1, 2000 and December 31, 2018. Elevated arsenic concentration in drinking water was recorded in Vladislavci, Cepin and Osijek area with values above the allowed maximum according to the EU standards (10 µg L-1) and as a result, arsenic levels in urine of the inhabitants were also elevated. Cumulative incidence for bladder cancer showed correlation between increased arsenic in water and urine in the areas affected by increased arsenic in water. Epidemiologic data suggest a conclusion that elevated arsenic could be considered at least as a cofounding factor for urinary tract cancer.

Effect of Prolonged Photoperiod on Light-Dependent Photosynthetic Reactions in Cannabis.

Industrial hemp is a fast-growing, short-day plant, characterized by high biomass yields and low demands for cultivation. To manipulate growth, hemp is usually cultivated under prolonged photoperiods or continuous light that could cause photooxidative damage and adjustments of photosynthetic reactions. To determine the extent of changes in photosynthetic response caused by prolonged light exposure, we employed chlorophyll a fluorescence measurements accompanied with level of lipid peroxidation (TBARS) and FT-IR spectroscopy on two Cannabis cultivars. Plants were grown under white (W) and purple (P) light at different photoperiods (16/8, 20/4, and 24/0). Our results showed diverse photosynthetic reactions induced by the different light type and by the duration of light exposure in two cultivars. The most beneficial condition was the 16/8 photoperiod, regardless of the light type since it brought the most efficient physiological response and the lowest TBARS contents suggesting the lowest level of thylakoid membrane damage. These findings indicate that different efficient adaptation strategies were employed based on the type of light and the duration of photoperiod. White light, at both photoperiods, caused higher dissipation of excess light causing reduced pressure on PSI. Efficient dissipation of excess energy and formation of cyclic electron transport around PSI suggests that P20/4 initiated an efficient repair system. The P24/0 maintained functional electron transport between two photosystems suggesting a positive effect on the photosynthetic reaction despite the damage to thylakoid membranes.

Contrasting Water Withholding Responses of Young Maize Plants Reveal Link Between Lipid Peroxidation and Osmotic Regulation Corroborated by Genetic Analysis.

Linking biochemistry and genetics of tolerance to osmotic stress is of interest for understanding plant adaptations to unfavorable conditions. The aims of this study were to investigate the variability in responses of panel of elite maize inbred lines to water withholding for stress-related traits through association study and to identify pathways linked to detected associations for better understanding of maize stress responses. Densely genotyped public and expired Plant Variety Protection Certificate (ex-PVP) inbred lines were planted in controlled conditions (16-h/8-h day/night, 25°C, 50% RH) in control (CO) and exposed to 10-day water withholding (WW). Traits analyzed were guaiacol peroxidase activity (GPOD), total protein content (PROT), lipid peroxidation (TBARS), hydrogen peroxide accumulation (H2O2), proline accumulation (proline), and current water content (CWC). Proline accumulation was found to be influenced by H2O2 and TBARS signaling pathways acting as an accumulation-switching mechanism. Most of the associations detected were for proline (29.4%) and TBARS (44.1%). Gene ontology (GO) enrichment analysis showed significant enrichment in regulation of integral membrane parts and peroxisomes along with regulation of transcription and polysaccharide catabolism. Dynamic studies involving inbreds with extreme phenotypes are needed to elucidate the role of this signaling mechanism in regulation of response to water deficit.

Physiological, Biochemical and Molecular Response of Different Winter Wheat Varieties under Drought Stress at Germination and Seedling Growth Stage.

Due to climate change in recent years, there has been an increasing water deficit during the winter wheat sowing period. This study evaluated six Croatian winter wheat varieties' physiological, biochemical, and molecular responses under two drought stress levels at the germination/seedling growth stage. Lipid peroxidation was mainly induced under both drought stress treatments, while the antioxidative response was variety-specific. The most significant role in the antioxidative response had glutathione along with the ascorbate-glutathione pathway. Under drought stress, wheat seedlings responded in proline accumulation that was correlated with the P5CS gene expression. Expression of genes encoding dehydrins (DHN5, WZY2) was highly induced under the drought stress in all varieties, while genes encoding transcription factors were differentially regulated. Expression of DREB1 was upregulated under severe drought stress in most varieties, while the expression of WRKY2 was downregulated or revealed control levels. Different mechanisms were shown to contribute to the drought tolerance in different varieties, which was mainly associated with osmotic adjustment and dehydrins expression. Identifying different mechanisms in drought stress response would advance our understanding of the complex strategies contributing to wheat tolerance to drought in the early growth stage and could contribute to variety selection useful for developing new drought-tolerant varieties.

Blood biomarkers in white stork (Ciconia ciconia) nestlings show different responses in several areas of Croatia.

White stork nestlings can provide quantitative data on the quality of the environment, as they are dependent on their parents that provide locally foraged food. Blood was sampled from the brachial vein (n = 109) and the sampling was performed in parallel with ringing during breeding season 2020 from five areas in eastern Croatia: Lonjsko polje, Jelas polje, Slavonski Brod-east, Podunavlje, and Donje Podravlje. In the present study, for the first time in Croatia, the following enzymatic biomarkers were assessed in white stork nestlings: activities of acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as nonenzymatic biomarkers: levels of glutathione (GSH) and reactive oxygen species (ROS). All endpoints were measured in two blood fractions: plasma and a postmitochondrial fraction (S9). Nestlings from Podunavlje and Donje Podravlje, areas known for intensive agriculture, showed lower AChE and CES activity when compared to the other investigated areas, indicating the presence of inhibitory xenobiotics. Higher oxidative stress was observed in Slavonski Brod-east, an area surrounded by metal and engineering industry, and Podunavlje compared to the other sampling areas. Hence, this study shows the impact of pollutants from the surrounding metal, petroleum, and agricultural industry might have on the biomarkers in white stork nestlings, which are often seen as early-warning signals.

Evaluation of Light-Dependent Photosynthetic Reactions in Reynoutria japonica Houtt. Leaves Grown at Different Light Conditions.

The Japanese knotweed (Reynoutria japonica Houtt.) is considered as one of the most aggressive and highly successful invasive plants with a negative impact on invaded habitats. Its uncontrolled expansion became a significant threat to the native species throughout Europe. Due to its extensive rhizome system, rapid growth, and allelopathic activity, it usually forms monocultures that negatively affect the nearby vegetation. The efficient regulation of partitioning and utilization of energy in photosynthesis enables invasive plants to adapt rapidly a variety of environmental conditions. Therefore, we aimed to determine the influence of light conditions on photosynthetic reactions in the Japanese knotweed. Plants were grown under two different light regimes, namely, constant low light (CLL, 40 µmol/m2/s) and fluctuating light (FL, 0-1,250 µmol/m2/s). To evaluate the photosynthetic performance, the direct and modulated chlorophyll a fluorescence was measured. Plants grown at a CLL served as control. The photosynthetic measurements revealed better photosystem II (PSII) stability and functional oxygen-evolving center of plants grown in FL. They also exhibited more efficient conversion of excitation energy to electron transport and an efficient electron transport beyond the primary electron acceptor QA, all the way to PSI. The enhanced photochemical activity of PSI suggested the formation of a successful adaptive mechanism by regulating the distribution of excitation energy between PSII and PSI to minimize photooxidative damage. A faster oxidation at the PSI side most probably resulted in the generation of the cyclic electron flow around PSI. Besides, the short-term exposure of FL-grown knotweeds to high light intensity increased the yield induced by downregulatory processes, suggesting that the generation of the cyclic electron flow protected PSI from photoinhibition.

Application of Non-Destructive Methods: Biomarker Assays in Blood of White Stork (Ciconia ciconia) Nestlings.

White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings' physiological condition, biomarkers can be measured in plasma and S9.

Lignin synthesis and accumulation in barley cultivars differing in their resistance to lodging.

Since lignin greatly affects stem strength, which is an important agronomical trait, understanding the relationship between lodging resistance and lignin synthesis is important in barley breeding and selection processes. The aim of the study was to reveal the connection between physiological aspects of lignin synthesis and genetic background of barley cultivars with different lodging phenotype. Three barley cultivars Astor, Scarlett and Jaran were compared by measuring lignin, cellulose and total soluble phenolics content, phenylalanine ammonia-lyase activity (PAL) and expression of cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) in three lower internodes at flowering and grain filling stage. To assess their genetic background simple sequence repeats (SSR) markers, connected to lodging resistance and plant height, were analyzed. Compared to lodging susceptible cultivars Scarlett and Jaran, a lodging resistant cultivar Astor revealed different dynamics of lignin synthesis and deposition, showing higher PAL activity and total soluble phenolics content as well as higher expression of CCR and CAD genes in the second internode at grain filling stage. Analysis of SSR markers associated with quantitative trait loci (QTL) for lodging resistance revealed that Astor discriminates from Scarlett and Jaran by marker Bmag337 connected with elongation of the second internode. Lignification process is under a strong influence of genotype and environmental factors which determine lignin synthesis dynamics and deposition of lignin in the cell walls of barley.

Cell-Type-Specific Modulation of Hydrogen Peroxide Cytotoxicity and 4-Hydroxynonenal Binding to Human Cellular Proteins In Vitro by Antioxidant Aloe vera Extract.

Although Aloe vera contains numerous bioactive components, the activity principles of widely used A. vera extracts are uncertain. Therefore, we analyzed the effects of genuine A. vera aqueous extract (AV) on human cells with respect to the effects of hydrogen peroxide (H2O2) and 4-hydroxynonenal (HNE). Fully developed A. vera leaves were harvested and analyzed for vitamin C, carotenoids, total soluble phenolic content, and antioxidant capacity. Furthermore, human cervical cancer (HeLa), human microvascular endothelial cells (HMEC), human keratinocytes (HaCat), and human osteosarcoma (HOS) cell cultures were treated with AV extract for one hour after treatment with H2O2 or HNE. The cell number and viability were determined using Trypan Blue, and endogenous reactive oxygen species (ROS) production was determined by fluorescence, while intracellular HNE⁻protein adducts were measured for the first time ever by genuine cell-based HNE⁻His ELISA. The AV extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq/g fresh weight) and cell-type-specific influence on the cytotoxicity of H2O2, as well as on endogenous production of ROS and HNE⁻protein adducts induced by HNE treatment, while AV itself did not induce production of ROS or HNE⁻protein adducts at all. This study, for the first time, revealed the importance of HNE for the activity principles of AV. Since HMEC cells were the most sensitive to AV, the effects of AV on microvascular endothelia could be of particular importance for the activity principles of Aloe vera extracts.

Effect of UVC radiation on mouse fibroblasts deficient for FAS-associated protein with death domain.

PURPOSE: Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell. MATERIALS AND METHODS: Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m(2)) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay. RESULTS: Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells. CONCLUSIONS: Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.

Response of Lemna minor L. to short-term cobalt exposure: The effect on photosynthetic electron transport chain and induction of oxidative damage.

The effect of two concentrations of cobalt (Co(2+)) on photosynthetic activity and antioxidative response in Lemna minor L. were assessed 24, 48 and 72h after the start of the exposure. Higher concentration of cobalt (1mM) induced growth inhibition while lower concentration (0.01mM) increased photosynthetic pigments content. Analysis of chlorophyll a fluorescence transients revealed high sensitivity of photosystem II primary photochemistry to excess of Co(2+) especially at the higher concentration where decreased electron transport beyond primary quinone acceptor QA(-) and impaired function of oxygen evolving complex (OEC) was observed. Due to impairment of OEC, oxygen production was decreased at higher Co(2+) concentration. Activity of superoxide dismutase was mainly inhibited while lipid peroxidation increased, at both concentrations, indicating that cobalt-induced oxidative damage after short exposure and moreover, susceptibility of the membranes in the cell to cobalt toxicity. Results obtained in this study suggest possible application of used parameters as tools in assessment of early damage caused by metals.