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Povzetek: The sensitivity of marine microalgae to metal contamination, particularly copper (Cu), is well recognized in ecotoxicology, providing key insights into predicting marine pollution impacts. This study examined the combined effects of Cu toxicity and salinity on the diatom Chaetoceros calcitrans under controlled laboratory conditions. Toxicity assays were conducted using two environmentally relevant Cu concentrations (2 and 5 μg L−1) and three salinity levels (33, 36, and 39) over 96 h, with cell responses analyzed via flow cytometry. Specific growth rate (SGR), chlorophyll fluorescence, fatty acid methyl ester (FAME) composition, polyunsaturated aldehydes (PUAs), and reactive oxygen species (ROS) production were evaluated. Results showed that SGR increased from 1.7 d−1 at salinity 33 to 1.9 d−1 at 39, indicating enhanced growth at higher salinity. However, Cu exposure significantly reduced SGR, particularly under 5 μg L−1 Cu at salinity 33, where SGR dropped to 0.86 d−1. ROS levels were highest under the combined low salinity (33) and 5 μg L−1 Cu treatment, suggesting strong oxidative stress. PUA profiles shifted with Cu and salinity, with stress-related compounds such as 2E,4E/Z-decadienal and 2E,4E/Z,7Z-decatrienal increasing at low salinity. Fatty acid analysis revealed marked rises in saturated fatty acids, especially palmitic acid (C16:0) and myristic acid (C14:0), under Cu exposure. In the Cu2/39 treatment, C16:0 reached 1.34 × 104 fmol cell−1 compared to 1.36 × 103 fmol cell−1 in the control. Monounsaturated fatty acids, notably palmitoleic acid (C16:1), also increased, reaching 7. 46 × 103 fmol cell−1 versus 1.71 × 103 fmol cell−1 in the control. Conversely, polyunsaturated fatty acids, including arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5 n-3), decreased significantly following Cu exposure. Overall, elevated salinity mitigated Cu-induced growth inhibition but intensified oxidative and biochemical stress, underscoring the complex, endpoint-specific responses of C. calcitrans to multiple environmental stressors.
Ključne besede: climate change, ecotoxicology, marine diatom, flow cytometry, lipids
Objavljeno v DiRROS: 11.06.2026; Ogledov: 340; Prenosov: 384
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Povzetek: Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are promising markers of vascular damage and endothelial regeneration potential. We focused on the detection of CECs and EPCs using flow cytometry with regard to analytical challenges and its suitability for routine testing. As part of a clinical validation, CECs and EPCs were measured in blood samples from 83 subjects with type 1 diabetes (T1DM), evaluating an adjuvant intervention with two different antidiabetic drugs, empagliflozin (N = 28) and semaglutide (N = 29). Both groups receiving adjuvant therapy were compared with the insulin-only group (N = 26) at two time points: before the start of therapy and after 12 weeks of adjuvant therapy. All three groups were comparable regarding demographic characteristics and concomitant risk factors. Absolute and relative endothelial cell count at baseline were low and comparable to those of healthy individuals. In the group receiving empagliflozin or semaglutide, a significant increase in EPC was observed after 12 weeks of treatment. We demonstrated that EPCs have the potential to serve as markers for monitoring the efficacy of adjuvant therapy in T1DM patients. However, before their implementation in clinical practice, the flow cytometry protocol for CEC and EPC identification and quantification must be standardized.
Ključne besede: flow cytometry, type 1 diabetes mellitus, endothelial progenitor cells
Objavljeno v DiRROS: 28.11.2025; Ogledov: 898; Prenosov: 405
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Povzetek: 3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.
Ključne besede: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks
Objavljeno v DiRROS: 16.07.2024; Ogledov: 1750; Prenosov: 919
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