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Toxics
2022 Feb 10;102:. doi: 10.3390/toxics10020083.
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Vanadium Toxicity Monitored by Fertilization Outcomes and Metal Related Proteolytic Activities in Paracentrotus lividus Embryos.
Chiarelli R
,
Martino C
,
Roccheri MC
,
Geraci F
.
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Metal pharmaceutical residues often represent emerging toxic pollutants of the aquatic environment, as wastewater treatment plants do not sufficiently remove these compounds. Recently, vanadium (V) derivatives have been considered as potential therapeutic factors in several diseases, however, only limited information is available about their impact on aquatic environments. This study used sea urchin embryos (Paracentrotus lividus) to test V toxicity, as it is known they are sensitive to V doses from environmentally relevant to very cytotoxic levels (50 nM; 100 nM; 500 nM; 1 µM; 50 µM; 100 µM; 500 µM; and 1 mM). We used two approaches: The fertilization test (FT) and a protease detection assay after 36 h of exposure. V affected the fertilization percentage and increased morphological abnormalities of both egg and fertilization envelope, in a dose-dependent manner. Moreover, a total of nine gelatinases (with apparent molecular masses ranging from 309 to 22 kDa) were detected, and their proteolytic activity depended on the V concentration. Biochemical characterization shows that some of them could be aspartate proteases, whereas substrate specificity and the Ca2+/Zn2+ requirement suggest that others are similar to mammalian matrix metalloproteinases (MMPs).
Figure 1. Effects of V exposure on the percentage of fertilization events in the sea urchin Paracentrotus lividus. Upper panel: images of representative eggs captured by light microscopy. Egg with normal morphology and normal fertilization membrane (A). Egg with normal morphology and abnormal fertilization membrane (B). Egg with abnormal morphology and normal fertilization membrane (C). Egg with abnormal morphology and abnormal fertilization membrane (D). Unfertilized egg with normal morphology (E). Unfertilized egg with abnormal morphology (F). Bar = 50 µm. Lower panel: histogram bars showing the percentage of the number of eggs with each morphology per total of eggs used in each treatment. % of eggs with normal morphology and normal fertilization membrane (a). % of eggs with normal morphology and abnormal fertilization membrane (b). % of eggs with abnormal morphology and normal fertilization membrane (c). % of eggs with abnormal morphology and abnormal fertilization membrane (d). % of unfertilized eggs with normal morphology (e). % of unfertilized eggs with abnormal morphology (f). The statistical significances was set to p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).
Figure 2. Proteolytic activities analyzed by gelatin substrate gel zimography. (A) Zimogram showing gelatinase bands in lysates of embryos at 36 h of growth from: control and V-treated embryos. M = protein molecular weight marker. (B) The pie charts display the percentages of each gelatinase activity (309, 225, 177, 79, 59, 34, 30, 25, and 22 kDa), for control and treated embryos after 36 h of development.
Figure 3. Relative gelatinase activities. The histograms show the percentage of relative gelatinase activity for each gelatinase in control and V-treated embryos after 36 h of development. All values were normalized with respect to the gelatinases activity of the control samples (fixed to 100%). The statistical significances were set to p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0005 (***).
Figure 4. Proteolytic activities analyzed by gelatin substrate gel zimography for control (C) and V-treated (V) embryos. The analysis was conducted in the absence (NA) or in the presence of a variety of protease inhibitors: Ethylenediaminetetraacetic acid (EDTA); ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA); 1,10 phenanthroline (1,10-Phe); Dithiothreitol (DTT); Phenylmethylsulfonyl fluoride (PMSF); pepstatin A (peps A).
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