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Int J Mol Sci
2022 Jun 02;2311:. doi: 10.3390/ijms23116239.
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Toxicity of Vanadium during Development of Sea Urchin Embryos: Bioaccumulation, Calcium Depletion, ERK Modulation and Cell-Selective Apoptosis.
Chiarelli R
,
Scudiero R
,
Memoli V
,
Roccheri MC
,
Martino C
.
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Vanadium toxicology is a topic of considerable importance as this metal is widely used in industrial and biomedical fields. However, it represents a potential emerging environmental pollutant because wastewater treatment plants do not adequately remove metal compounds that are subsequently released into the environment. Vanadium applications are limited due to its toxicity, so it is urgent to define this aspect. This metal is associated with sea urchin embryo toxicity as it perturbs embryogenesis and skeletogenesis, triggering several stress responses. Here we investigated its bioaccumulation and the correlation with cellular and molecular developmental pathways. We used cytotoxic concentrations of 1 mM and 500 μM to perform quantitative analyses, showing that vanadium accumulation interferes with calcium uptake during sea urchin development and provokes a disruption in the biomineralization process. At the end of the whole treatment, the accumulation of vanadium was about 14 and 8 μg for embryos treated respectively with 1 mM and 500 μM, showing a dose-dependent response. Then, we monitored the cell signaling perturbation, analyzing key molecular markers of cell survival/cell death mechanisms and the DNA fragmentation associated with apoptosis. This paper clarifies vanadium's trend to accumulate directly into embryonic cells, interfering with calcium uptake. In addition, our results indicate that vanadium can modulate the ERK pathway and activate a cell-selective apoptosis. These results endorse the sea urchin embryo as an adequate experimental model to study metal-related cellular/molecular responses.
Figure 1. Pictures of representative embryos at 36 h of development/treatment. Control embryo (A), 1 mM V-treated embryo (B), 500 μM V-treated embryo (C). Bar: 100 μm.
Figure 2. Amount of V and Ca incorporated during the time after 12, 18, 24, 30, 36 and 42 h of development/treatment. Embryos were cultured in 1 mM or 500 μM of V. V (A) and Ca (B) content were detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), determining the metal quantity in about 250,000 embryos. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD).
Figure 3. Immunoblotting detection and quantitative analysis for pERK and ERK 1/2. (A) Total lysates of control and V-treated (1 mM, 500 μM) embryos after 24, 30, 36 and 42 h of development/treatment. Actin was used as a loading control. Histograms show the densitometric analysis of bands identified for (B) pERK and (C) ERK 1/2. Relative protein expression, reported as arbitrary units, was calculated as the band density ratio to that of actin. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Data were analyzed by one-way ANOVA. Treatments with the same lowercase letter do not differ (Tukey HSD).
Figure 4. Immunoblotting detection and quantitative analysis for CHOP 10/GADD 153 and cleaved caspase 7. (A) Total lysates of control and V-treated (1 mM, 500 μM) embryos after 24, 30, 36 and 42 h of development/treatment. Actin was used as a loading control. Histograms show the densitometric analysis of bands identified for (B) CHOP-10/GADD 153 and (C) cleaved caspase 7. Relative protein expression, reported as arbitrary units, was calculated as the band density ratio to that of actin. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Data were analyzed by one-way ANOVA. Treatments with the same lowercase letter do not differ (Tukey HSD).
Figure 5. Fluorescent TUNEL assay and densitometric analysis. Pictures of demonstrative embryos at 24, 30, 36 and 42 h of development/treatment. DNA fragmentation (A1âN1). Nuclei marked with propidium iodide (A2âN2). Merge of both signals (A3âN3). Control embryos (A1âA3,D1âD3,H1âH3,K1âK3); 1 mM V-treated embryos (B1âB3,E1âE3,I1âI3,L1âL3); 500 μM V-treated embryos (C1âC3,F1âF3,J1âJ3,M1âM3). Positive control embryo at 42 h of development (G1âG3). Negative control embryo at 42 h of development (N1âN3). Bar = 100 μm. Histograms showing data related to the quantitative analysis of fluorescence from apoptotic DNA. Experiments were performed in triplicate and data are expressed as means ± standard deviation (n = 3 ± SD). Data were analyzed by one-way ANOVA. Treatments with the same lowercase letter do not differ (Tukey HSD).
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