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Toxicol Sci
2016 Jun 01;1512:419-33. doi: 10.1093/toxsci/kfw053.
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First Morphological and Molecular Evidence of the Negative Impact of Diatom-Derived Hydroxyacids on the Sea Urchin Paracentrotus lividus.
Varrella S
,
Romano G
,
Ruocco N
,
Ianora A
,
Bentley MG
,
Costantini M
.
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Oxylipins (including polyunsaturated aldehydes [PUAs], hydoxyacids, and epoxyalcohols) are the end-products of a lipoxygenase/hydroperoxide lyase metabolic pathway in diatoms. To date, very little information is available on oxylipins other than PUAs, even though they represent the most common oxylipins produced by diatoms. Here, we report, for the first time, on the effects of 2 hydroxyacids, 5- and 15-HEPE, which have never been tested before, using the sea urchin Paracentrotus lividus as a model organism. We show that HEPEs do induce developmental malformations but at concentrations higher when compared with PUAs. Interestingly, HEPEs also induced a marked developmental delay in sea urchin embryos, which has not hitherto been reported for PUAs. Recovery experiments revealed that embryos do not recover following treatment with HEPEs. Finally, we report the expression levels of 35 genes (involved in stress, development, differentiation, skeletogenesis, and detoxification processes) to identify the molecular targets affected by HEPEs. We show that the 2 HEPEs have very few common molecular targets, specifically affecting different classes of genes and at different times of development. In particular, 15-HEPE switched on fewer genes than 5-HEPE, upregulating mainly stress-related genes at a later pluteus stage of development. 5-HEPE was stronger than 15-HEPE, targeting 24 genes, mainly at the earliest stages of embryo development (at the blastula and swimming blastula stages). These findings highlight the differences between HEPEs and PUAs and also have important ecological implications because many diatom species do not produce PUAs, but rather these other chemicals are derived from the oxidation of fatty acids.
FIG. 1. Cleavage inhibition in sea urchin embryos following decadienal (at the concentrations of 1.3, 1.6, 2.6, 3.3, 5.3, 6.6, 8.2, 9.9, 11.8, 13.1, 14.5, 15.8, 17.1, 18.4, and 19.7 μM; black line; data from Romano et al., 2010) and 5- and 15-HEPE (at the concnetrations of 6, 7, 8, 9, 10, 15, 30, 50, 70 and 90 μM; blue line) treatments. Control is reported as 0 μM concentration. Abbreviation: HEPE, hydroxyacids.
FIG. 2. Abnormal and delayed embryos after HEPEs treatments. Top panel: percentage (%) of abnormal and delayed embryos when Paracentrotus lividus newly fertilized eggs were exposed to different concentrations of the polyunsaturated aldehydes decadienal (1.32, 2.63, 3.95, 5.26, and 6.58 μM; Romano et al., 2010) and 5- and 15-HEPEs (6, 7, 8, 9, 10, 15, and 30 μM) at 48 h post fertilization (hpf). Significant differences (mean ± SD) compared with the control (4.3 ± 0.8 abnormal embryos; 3.3 ± 1.0 delayed embryos, data not shown): *P < .05, **P < .01, ***P < .001. One-way ANOVA (P < .05) with Tukey’s multiple comparison test. Experiments were conducted in triplicate using 3 egg groups collected from 3 different females. Bottom panel (A) control embryos (in filtered sea water) without HEPEs) at 48 hpf; (B) delayed embryos observed in samples treated with HEPEs at the concentrations from 6 to 10 µM and (C) delayed embryos observed in samples treated with HEPEs at 15 and 30 µM at 48 hpf. HEPE, hydroxyacids.
FIG. 3. Real-time quantitative PCR (qPCR) at blastula stage. Histograms show the differences in expression levels of analyzed genes belonging to different functional classes (stress, detoxification, development and differentiation and skeletogenesis), followed by real time qPCR. Paracentrotus lividus embryos were grown in the presence of 15-HEPE and 5-HEPE at 7.0 µM and collected at 5 h post fertilization. Data are reported as a fold difference compared with control (mean ± SD) embryos in sea water without HEPEs. Fold differences greater than ±2 (see dotted horizontal guidelines at values of 2 and −2) were considered significant. Abbreviation: HEPE, hydroxyacids.
FIG. 4. Real-time quantitative PCR (qPCR) a swimming blastula stage. Histograms show the differences in expression levels of analyzed genes, followed by real-time qPCR. Paracentrotus lividus embryos were grown in the presence of 7.0 µM of 15-HEPE and 5-HEPE and collected at 9 h post fertilization (for further details see also the legend to Figure 3). Abbreviation: HEPE, hydroxyacids.
FIG. 5. Real-time quantitative PCR (qPCR) at prism stage. Histograms show the differences in expression levels of analyzed genes, followed by real-time qPCR. Paracentrotus lividus embryos were grown in the presence of 7.0 µM of 15-HEPE and 5-HEPE and collected at 24 h post fertilization (for further details see also the legend to the Figure 3). Abbreviation: HEPE, hydroxyacids.
FIG. 6. Real-time quantitative PCR (qPCR) at pluteus stage. Histograms show the differences in expression levels of analyzed genes, followed by real-time qPCR. Paracentrotus lividus embryos were grown in the presence of 7.0 µM of 15-HEPE and 5-HEPE and collected at 48 h post fertilization (for further details see also the legend to the Figure 3). Abbreviation: HEPE, hydroxyacids.
FIG. 7. Synopsis of real time qPCR experiments. Synopsis of the patterns of up- and downregulation of different classes of genes in the sea urchin, Paracentrotus lividus, in the presence of HEPEs. PUAs decadienal, heptadienal, and octadienal are reported for the sake of comparison (Varrella et al., 2014). Abbreviation: HEPE, hydroxyacids.
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