The influence of copper-doped mesoporous bioactive nanospheres on the temperature rise during polymerization, polymer cross-linking density, monomer release and embryotoxicity of dental composites.

OBJECTIVES: Composites with copper-doped mesoporous bioactive nanospheres (Cu-MBGN) were developed to prevent secondary caries by imparting antimicrobial and ion-releasing/remineralizing properties. METHODS: Seven experimental composites containing 1, 5 or 10 wt% Cu-MBGN, the corresponding inert controls (silica) and bioactive controls (bioactive glass 45S5) were prepared. The temperature rise during light curing, cross-linking density by ethanol softening test, monomer elution and their potential adverse effects on the early development of zebrafish Danio rerio was investigated. RESULTS: Materials combining Cu-MBGN and silica showed the highest resistance to ethanol softening, as did the bioactive controls. Cu-MBGN composites showed significant temperature rise and reached maximum temperature in the shortest time. Bisphenol A was not detected, while bis-GMA was found only in the control materials and TEGDMA in the eluates of all materials. There was no increase in zebrafish mortality and abnormality rates during exposure to the eluates of any of the materials. CONCLUSIONS: The composite with 5 wt% Cu-MBGN combined with nanosilica fillers showed the lowest ethanol softening, indicating the polymer's highest durability and cross-linking density. Despite the TEGDMA released from all tested materials, no embryotoxic effect was observed.

Intra-Species Variations of Bioactive Compounds of Two Dictyota Species from the Adriatic Sea: Antioxidant, Antimicrobial, Dermatological, Dietary, and Neuroprotective Potential.

The marine environment has a significant impact on life on Earth. Organisms residing in it are vital for the ecosystem but also serve as an inexhaustible source of biologically active compounds. Herein, the biodiversity of two brown seaweeds, Dictyota dichotoma and Dictyota fasciola from the Adriatic Sea, was evaluated. The aim of the study was the determination of differences in compound composition while comparing their activities, including antioxidant, antimicrobial, and enzyme inhibition, in connection to human digestion, dermatology, and neurological disorders. Chemical analysis revealed several terpenoids and steroids as dominant molecules, while fucoxanthin was the main identified pigment in both algae. D. dichotoma had higher protein, carbohydrate, and pigment content. Omega-6 and omega-3 fatty acids were identified, with the highest amount of dihomo-γ-linolenic acid and α-linolenic acid in D. dichotoma. Antimicrobial testing revealed a dose-dependent inhibitory activity of methanolic fraction against Escherichia coli and Staphylococcus aureus. Moderate antioxidant activity was observed for both algae fractions, while the dietary potential was high, especially for the D. fasciola dichloromethane fraction, with inhibition percentages of around 92% for α-amylase and 57% for pancreatic lipase at 0.25 mg/mL. These results suggest that Dictyota species might be a potent source of naturally derived agents for obesity and diabetes.

Fish liver damage related to the wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) continuously release a complex mixture of municipal, hospital, industrial, and runoff chemicals into the aquatic environment. These contaminants are both legacy contaminants and emerging-concern contaminants, affecting all tissues in a fish body, particularly the liver. The fish liver is the principal detoxifying organ and effects of consistent pollutant exposure can be evident on its cellular and tissue level. The objective of this paper is thus to provide an in-depth analysis of the WWTP contaminants' impact on the fish liver structure, physiology, and metabolism. The paper also gives an overview of the fish liver biotransformation enzymes, antioxidant enzymes, and non-enzymatic antioxidants, their role in metabolizing xenobiotic compounds and coping with oxidative damage. Emphasis has been placed on highlighting the vulnerability of fish to xenobiotic compounds, and on biomonitoring of exposed fish, generally involving observation of biomarkers in caged or native fish. Furthermore, the paper systematically assesses the most common contaminants with the potential to affect fish liver tissue.

In Vivo and In Vitro Antioxidant Activity of Less Polar Fractions of Dasycladus vermicularis (Scopoli) Krasser 1898 and the Chemical Composition of Fractions and Macroalga Volatilome.

The present research is a comprehensive investigation of Dasycladus vermicularis (Scopoli) Krasser 1898 from the Adriatic Sea (Croatia) regarding volatilome-volatile organic compounds (VOCs, mostly nonpolar compounds) and less polar nonvolatile compounds for the first time. Headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were used showing the great volatilome variability among fresh (HS-FrDV and HD-FrDV) and dried (HS-DrDV and HD-DrDV) samples after GC-MS analysis. Aromatic aldehydes were dominant in both fresh and air-dried HS samples with benzaldehyde as the most abundant in fresh samples and decreasing 2.7-3.7 times after drying together with 2-phenylbut-2-enal that was not present after drying. Aliphatic compounds (unsaturated hydrocarbons in HS-FrDV; saturated hydrocarbons in HS-DrDV) were also present. C11-hydrocarbons (dictyopterpene C' and dictyopterpene D') were detected in HS-FrDV. (E)-Phytol was the most dominant compound in HD-FrDV and HD-DrDV. Diterpene alcohols (cembra-4,7,11,15-tetraen-3-ol and (Z)-falcarinol) and sesquiterpene alcohol, cubenol, were dominant in HD-FrDV, and their abundance decreased after drying. C13-norisoprenoides (α-ionone and ß-ionone) increased after drying. Aliphatic compounds were present in both HD-FrDV and HD-DrDV samples. The less polar nonvolatile compounds in the obtained fractions F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C55H74N4O5-7), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represented the majority of identified compounds. By implementing both in vitro and in vivo assays for antioxidant activity determination, F3 showed a higher activity than F4. Inhibitory concentrations (IC50) for F3 and F4 were 498.00 ± 0.01 µg/mL and 798.00 ± 0.81 µg/mL, respectively, while a 1.5-fold reduction in the ROS level was observed after pre-treatment of zebrafish larvae with 45 µg/mL of F3.

Bioprospecting of Less-Polar Fractions of Ericaria crinita and Ericaria amentacea: Developmental Toxicity and Antioxidant Activity.

Ericaria crinita and Ericaria amentacea from the Adriatic Sea (Croatia) were investigated with respect to the presence of less-polar compounds for the first time after fractionation by solid-phase extraction (SPE). The composition of less-polar fractions of freeze-dried E. crinita (FdEc) and E. amentacea (FdEa) were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). The major identified compounds were: amides of higher aliphatic acids (palmitoleamide, linoleamide, palmitamide, oleamide and erucamide) and related compounds, carotenoid (fucoxanthin), chlorophyll derivatives (pheophytin a and b and their derivatives) and higher terpenes (loliolide, isoamijiol with its oxidation product), ß-stigmasterol and (3ß,6α)-14-methylergosta-8,24(28)-diene-3,6-diol). The toxic effects observed on the less-polar fractions obtained from Ericaria species on zebrafish Danio rerio embryos could be associated with the high abundance of all five detected amides. The antioxidant activity of the fractions was evaluated by means of five independent assays, including the reduction of the radical cation (ABTS), the oxygen radical absorbance capacity (ORAC), ferric-reducing antioxidant power (FRAP), the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay and the Folin-Ciocalteu method. A higher antioxidant activity of E. amentacea in comparison to that of the E. crinita fractions was found with IC50 concentrations of 0.072 and 1.177 mg/mL, respectively. The correlation between the activity and the chemical composition revealed that the synergistic effect of different compounds impacted their antioxidant response.

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00 mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00 mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00 mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Supercritical Carbon Dioxide Extraction of Four Medicinal Mediterranean Plants: Investigation of Chemical Composition and Antioxidant Activity.

With everyday advances in the field of pharmaceuticals, medicinal plants have high priority regarding the introduction of novel synthetic compounds by the usage of environmentally friendly extraction technologies. Herein, a supercritical CO2 extraction method was implemented in the analysis of four plants (chamomile, St. John's wort, yarrow, and curry plant) after which the non-targeted analysis of the chemical composition, phenolic content, and antioxidant activity was evaluated. The extraction yield was the highest for the chamomile (5%), while moderate yields were obtained for the other three plants. The chemical composition analyzed by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) demonstrated extraction of diverse compounds including terpenes and terpenoids, fatty acids, flavonoids and coumarins, functionalized phytosterols, and polyphenols. Voltammetry of microfilm immobilized on a glassy carbon electrode using square-wave voltammetry (SWV) was applied in the analysis of extracts. It was found that antioxidant activity obtained by SWV correlates well to 1,1-diphenyl-2-picrylhidrazine (DPPH) radical assay (R2 = 0.818) and ferric reducing antioxidant power (FRAP) assay (R2 = 0.640), but not to the total phenolic content (R2 = 0.092). Effective results were obtained in terms of activity showing the potential usage of supercritical CO2 extraction to acquire bioactive compounds of interest.

Less Polar Compounds and Targeted Antioxidant Potential (In Vitro and In Vivo) of Codium adhaerens C. Agardh 1822.

Codium adhaerens from the Adriatic Sea (Croatia) was comprehensively investigated regarding less polar compounds for the first time. Although there are several phytochemical studies on C. adhaerens from other regions, this is the first report on volatile organic compounds (VOCs) from fresh (FrCa) and air-dried (DrCa) samples. The novelty is also related to its targeted antioxidant potential in vitro and in vivo. The main aims were to: (a) identify and compare VOCs of FrCa and DrCa obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD); (b) determine fatty acid (FA) composition of freeze-dried sample (FdCa); (c) determine the composition of less polar fractions of FdCa by high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI-HRMS); and (d) comprehensively evaluate the antioxidant activity of the fractions by four in vitro assays and in vivo zebrafish model (including embryotoxicity). Significant changes of VOCs were found after air drying. ω6 FAs were present in higher content than ω3 FAs indicating C. adhaerens as a good source of dietary polyunsaturated FAs. The results obtained in vivo correlate well with in vitro methods and both fractions exerted similar antioxidative responses which is in agreement with the high abundance of present biomolecules with known antioxidant properties (e.g., fucoxanthin, pheophytin a, and pheophorbide a). These results suggest that C. adhaerens might be a potent source of natural antioxidants that could be further used in the research of oxidative stress-related diseases.

Bioprospecting of Less-Polar Constituents from Endemic Brown Macroalga Fucus virsoides J. Agardh from the Adriatic Sea and Targeted Antioxidant Effects In Vitro and In Vivo (Zebrafish Model).

The endemic brown macroalga Fucus virsoides J. Agardh from the Adriatic Sea was in the focus of the present research. The volatiles of fresh (FrFv) and air-dried (DrFv) samples of F. virsoides obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were analyzed by gas chromatography equipped with flame ionization detector and mass spectrometry (GC-FID/MS). The major HS-FrFv compound was pentadecane (61.90-71.55%) followed by pentadec-1-ene (11.00-7.98%). In HS-DrFv, pentadec-1-ene was not present, and few lower aliphatic compounds appeared, as well as benzaldehyde and benzyl alcohol. In HD-FrFv, particularly abundant were alkenes (such as pentadec-1-ene (19.32%), or (E)-pentadec-7-ene (8.35%)). In HD-DrFv, more oxidation products were present (e.g., carbonyl compounds such as tridecanal (18.51%)). The fatty acids profile of freeze-dried sample (FdFv) after conversion to methyl esters was determined by GC-FID, and oleic acid was dominant (42.28%), followed by arachidonic acid (15.00%). High-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (HPLC-ESI-HRMS) was used for the screening of less polar fractions (F3 and F4) of F. virsoides. Mono- and diglycerides of stearic, palmitic, oleic, and arachidonic acids were found. Terpenoids and steroids comprised the compounds C20H30(32)O2 and C29H48O(2). Among carotenoids, fucoxanthin was identified. Chlorophyll derivatives were also found (C55H74(72)N4O(5-7)), dominated by pheophytin a. The antioxidant activity of the fractions was investigated by in vitro assays (oxygen radical absorbance capacity (ORAC), reduction of radical cation (ABTS•+), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay, and ferric reducing antioxidant power (FRAP)) and by in vivo zebrafish model (along with fish embryotoxicity). In vitro experiments proved good radical scavenging abilities of F3 and F4 fractions, which were additionally supported by the protective effect against hydrogen peroxide-induced oxidative stress in zebrafish embryos.

Bioprospecting of Coralline Red Alga Amphiroa rigida J.V. Lamouroux: Volatiles, Fatty Acids and Pigments.

Due to the lack of phytochemical composition data, the major goals of the present study on Amphiroa rigida J.V. Lamouroux were to: (a) investigate and compare volatilome profiles of fresh and air-dried samples obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) followed by gas chromatography and mass spectrometry (GC/MS) analysis; (b) determine fatty acids profile by gas chromatography with flame ionization detector (GC-FID); (c) obtain the pigment profiles of semipurified extracts by high performance liquid chromatography (HPLC) and (d) evaluate the antioxidant and antimicrobial activities of its less polar fractions. The comparison of headspace of fresh (FrAr) and air-dried (DrAr) samples revealed many similarities regarding the presence and abundance of the major (heptadecane and pentadecane) and minor compounds. The hydrodistillate (HD) of DrAr profile was quite different in comparison to HD-FrAr. The predominant compound in HD-FrAr was (E)-phytol. In HD-DrAr, its percentage was approximately one-half reduced, but the abundance of its degradation product phytone and of unsaturated and oxygenated compounds increased indicating more intense fatty acid decomposition and oxidation during drying. The fatty acid determination revealed that the most dominant was palmitic acid (42.86%) followed by eicosapentaenoic acid (19.14%) and stearic acid (11.65%). Among the pigments, A. rigida contained fucoxanthin (0.63 mg g- 1 of dry fraction), lutein (5.83 mg g- 1), ß-carotene (6.18 mg g-1) and chlorophyll a (13.65 mg g-1). The analyzed less polar fractions of A. rigida exhibited antioxidant scavenging activity with diammonium salt of 2,2'-azino-bis (3-ethylbenzthiazolin-6-yl) sulfonic acid (ABTS) assay up to 3.87 mg g-1 trolox equivalents (TE), and with the oxygen radical absorbance capacity (ORAC) assay up to 825.63 µmol g-1 TE (with carotenoids as the major contributors).

Utilization of the zebrafish model to unravel the harmful effects of biomass burning during Amazonian wildfires.

Amazonian wildfires in 2019 have raised awareness about rainforest burning due to increased emissions of particulate matter and carbon. In the context of these emissions, by-products of lignin thermal degradation (i.e. methoxyphenols) are often neglected. Methoxyphenols entering the atmosphere may form intermediates with currently unknown reaction mechanisms and toxicity. This study for the first time provides a comprehensive insight into the impact of lignin degradation products [guaiacol, catechol], and their nitrated intermediates [4-nitrocatechol, 4,6-dinitroguaiacol, 5-nitroguaiacol] on zebrafish Danio rerio. Results revealed 4-nitrocatechol and catechol as the most toxic, followed by 4,6DNG > 5NG > GUA. The whole-organism bioassay integrated with molecular modeling emphasized the potential of methoxyphenols to inhibit tyrosinase, lipoxygenase, and carbonic anhydrase, consequently altering embryonic development (i.e. affected sensorial, skeletal, and physiological parameters, pigmentation formation failure, and non-hatching of larvae). The whole-organism bioassay integrated with in silico approach confirmed the harmful effects of lignin degradation products and their intermediates on aquatic organisms, emphasizing the need for their evaluation within ecotoxicity studies focused on aquatic compartments.

Seasonal antioxidant and biochemical properties of the Northern Adriatic Pecten jacobaeus.

The present work is the first study of Mediterranean scallop (Pecten jacobaeus) biochemical properties, antioxidant defenses, and free radical scavengers during the yearly seasons in the Northern Adriatic, off Istria. Scallop nutrient reserves (glucose, triglyceride, and cholesterol) in four tissues under examination were positively correlated and were predominant in digestive gland and gonad. The muscle energy maxima were in correlation with the maximum fall gonosomatic index (GSI), when diatoms and coccolithophorids thrive. The decrease of GSI in summer might be related to the spawning or resorption of gametes. Summer also revealed elevated levels of glucose in gonad and digestive gland, while muscle glucose and cholesterol significantly varied in spring vs. winter samples. In relation to the diatom seasonal abundance, carotenoids, namely astaxanthin peaks were found in digestive gland, which, being stimulators of calcium transport over cell membranes, could have contributed to the high digestive gland levels of calcium in winter. In winter, total antioxidative status (TAS) of scallop tissues was 3-fold higher than in other seasons, particularly in digestive gland, having a significant correlation with magnesium, a regulatory tool in oxidative processes. The winter maxima of TAS and thiobarbituric acid reactive substances TBARS in relation to summer maxima of glutathione peroxidase and superoxide dismutase in digestive glands indicate to a decrease in antioxidant defense during cold months, and are related to the accumulation of lipid peroxidation products (such as malondialdehyde) in digestive gland of scallops. Although the increased susceptibility to oxidative stress could be attributed to winter temperature, other factors such as the gonad maturation, availability of food supply, and salinity might counteract that effect. The seawater alterations of salinity, temperature and water quality are in relation to the river Po influx, which is very likely to influence the physiological and biochemical responses of scallops in the Northern Adriatic.

Electrochemistry as a screening method in determination of carotenoids in crustacean samples used in everyday diet.

Electrochemistry of carotenoids has attracted a lot of interest because it provides an understanding of their oxidative properties. We report the application of electrochemistry in the analysis of carotenoids. Voltammetry of microdroplets immobilized on paraffin impregnated graphite electrode in 0.1 mol dm-3 HClO4 and KNO3 aqueous electrolytes using square-wave voltammetry was applied. Previous studies have shown that carotenoids undergo complex oxidation process when characterized in aqueous media. In this research, the electrooxidation of carotenoid astaxanthin was confirmed. The obtained response allowed the development of an electroanalytical method with a limit of detection of 15.77 µmol dm-3, the limit of quantification of 47.80 µmol dm-3 and acceptable relative standard deviations for current (3.69%) and potential (0.41%). Extraction using DMSO and acetone has shown to be appropriate for voltammetric analysis. Astaxanthin content was determined electrochemically in shrimp and soft-shell crab samples (20.63 and 6.59 µg g-1, respectively), yielding recoveries above 90%.