Teratogenic effects of diatom metabolites on sea urchin Paracentrotus lividus embryos

Article abstract of DOI:10.3390/md8040950

The diatom-derived polyunsaturated aldehydes (PUAs), 2-trans,4-trans- decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, as well as tridecanal were tested on early and later larval development in the sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant diatom polyunsaturated fatty acids (PUFAs) on development, in particular 5,8,11,14,17-eicosapentaenoic acid (EPA), one of the main precursors of diatom PUAs, as well as 4,7,10,13,16,19- docosahexaenoic acid (DHA), 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 6,9,12-octadecatrienoic acid (γ-linolenic acid) and 9,12-octadecadienoic acid (linoleic acid). PUAs blocked sea urchin cell cleavage in a dose dependent manner and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 μM with heptadienal, 16.13 μM with octadienal, 11.47 μM with octatrienal and 5.26 μM with decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at much higher concentrations compared to PUAs (331 μM for EPA and 181 μM for stearidonic acid). Sub-lethal concentrations of decadienal (1.32-5.26 μM) delayed development of embryos and larvae which showed various degrees of malformations depending on the concentrations tested. Sub-lethal concentrations also increased the proportion of TUNEL-positive cells indicating imminent death in embryos and larvae. Using decadienal as a model PUA, we show that this aldehyde can be detected spectrophotometrically for up to 14 days in f/2 medium.

Full text of DOI:10.3390/md8040950

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Mar. Drugs 2010, 8, 950-967; doi:10.3390/md8040950
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Teratogenic Effects of Diatom Metabolites on Sea Urchin
Paracentrotus lividus Embryos
Giovanna Romano *, Antonio Miralto and Adrianna Ianora
Stazione Zoologica Anton Dohrn, Villa Comunale 80121 Napoli, Italy;
E-Mails: antonio.miralto@szn.it (A.M.); adrianna.ianora@szn.it (A.I.)
* Author to whom correspondence should be addressed; E-Mail: giovanna.romano@szn.it;
Tel.: +39 081 5833430; Fax: +39 081 7641355.
Received: 24 February 2010; in revised form: 6 March 2010 / Accepted: 24 March 2010 /
Published: 30 March 2010
Abstract: The diatom-derived polyunsaturated aldehydes (PUAs), 2-trans,4-trans-
decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-
heptadienal, as well as tridecanal were tested on early and later larval development in the
sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant
diatom polyunsaturated fatty acids (PUFAs) on development, in particular 5,8,11,14,17-
eicosapentaenoic acid (EPA), one of the main precursors of diatom PUAs, as well as
4,7,10,13,16,19-docosahexaenoic acid (DHA), 6,9,12,15-octadecatetraenoic acid
(stearidonic acid), 6,9,12-octadecatrienoic acid (γ-linolenic acid) and 9,12-octadecadienoic
acid (linoleic acid). PUAs blocked sea urchin cell cleavage in a dose dependent manner
and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 µM
with heptadienal, 16.13 µM with octadienal, 11.47 µM with octatrienal and 5.26 μM with
decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at
much higher concentrations compared to PUAs (331 μM for EPA and 181 μM for
stearidonic acid). Sub-lethal concentrations of decadienal (1.32–5.26 μM) delayed
development of embryos and larvae which showed various degrees of malformations
depending on the concentrations tested. Sub-lethal concentrations also increased the
proportion of TUNEL-positive cells indicating imminent death in embryos and larvae.
Using decadienal as a model PUA, we show that this aldehyde can be detected
spectrophotometrically for up to 14 days in f/2 medium.

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Keywords: diatom; oxylipins; sea urchin; fatty acids; development arrest; apoptosis;
TUNEL; teratogens
1. Introduction
Diatoms are the dominant photosynthetic organisms in the world’s oceans, accounting for up to
40% of annual productivity at sea [1] and 25% of global carbon-fixation [2]. These microscopic,
unicellular algae have traditionally been considered as a good food for plankton primary consumers,
consisting mainly of small crustacean copepods that dominate the zooplankton, and in the transfer of
carbon to higher trophic levels including important fisheries. However, their beneficial role in
supporting marine food chains has been challenged with the discovery that some diatom species
produce polyunsaturated aldehydes (PUAs) having cytotoxic and antitumoral activity ([3] onwards).
PUAs are the end-products of a lipoxygenase/hydroperoxide lyase metabolic pathway [4] initiated by
damage to algal cells, as occurs through grazing by predators. Cell damage activates lipase enzymes,
which liberate polyunsaturated fatty acids (PUFAs) from cell membranes that are immediately
oxidized and cleaved within seconds to form PUAs and a plethora of other metabolites collectively
termed oxylipins [5,6] (Figure 1). The specific type and quantity of oxylipins produced differs between
species and strains due to a variety of precursor PUFAs and enzymes [4–7], with variable effects on
zooplankton grazers [8].
Oxyplins, and PUAs in particular, have important biological and biochemical properties including
the disruption of gametogenesis, gamete functionality, fertilization, embryonic mitosis, and larval
fitness and competence [9]. Although the effects of such toxins are less catastrophic than those
inducing poisoning and death of predators, they are none-the-less insidious occurring through
abortions, birth defects and reduced larval survivorship. Such antiproliferative compounds may
discourage herbivory by sabotaging future generations of grazers, thereby allowing diatom blooms to
persist when grazing pressure would otherwise have caused them to crash.
Diatom blooms are a potential energy source for benthic organisms as well, but the sinking of
diatoms to the sediment at the end of the bloom may have negative consequences on reproductive
activity in residing populations. Previous studies on benthic invertebrates have shown that PUAs
inhibit fertilization processes, reduce larval fitness and induce teratogenesis in several broadcast-
spawning species (see [9] for a review). For example, sea urchin gametes incubated in the diatom PUA
decadienal showed impaired fertilization success due to inhibition of both sperm motility [10,11] and
pronuclear fusion [12]. Arrest of cell cleavage has been reported by various authors in both
Paracentrotus lividus and Sphaerechinus granularis eggs treated with decadienal [3,13,14]. At
concentrations higher than the dose required to arrest cell cleavage progression, decadienal was found
to induce apoptotic events in P. lividus embryos as demonstrated by means of a suite of techniques
including the evaluation of caspase-3-like protease activity [15]. In addition, Hansen and co-workers
[12] have demonstrated that decadienal is able to inhibit tubulin polymerization, DNA synthesis and
cyclin B/Cdk1 kinase activity, leading to the arrest of the cell cycle in S. granularis early embryos.

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Here we compare the effects of different diatom-derived PUAs, namely 2-trans,4-trans-decadienal,
2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, and the saturated
aldehyde tridecanal [16,17] on early and later larval development in the sea urchin Paracentrotus
lividus. Apoptosis induction in the pluteus stage after chronic exposure to decadienal is evaluated for
the first time by means of TUNEL staining. Since it has been suggested that free PUFAs may
negatively affect reproductive success in sea urchins [18], we also tested the effect of some of the more
abundant diatom PUFAs on sea urchin development, in particular 5,8,11,14,17,-eicosapentaenoic acid
(EPA), one of the main precursors of diatom oxylipins, as well as 4,7,10,13,16,19-docosahexaenoic
acid (DHA), 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 6,9,12-octadecatrienoic acid
(γ-linolenic acid) and 9,12-octadecadienoic acid (linoleic acid). The deleterious teratogenic effects of
PUAs on sea urchin embryos are compared with those on other marine invertebrates.
Figure 1. Biosynthetic sketch for the synthesis of oxylipins from fatty acids via
lipoxygenase enzymes (LOX) in marine diatoms. R1 represent methyl terminal part and R2
the carboxylic end of C16 or C20 fatty acid precursors. R1 and R2 may differ in terms of
length and degree of unsaturation depending on the position of O2 addition by LOX in the
fatty acid molecule (modified from Fontana et al. [6]).
R1
R2
fatty acid
O2
LOX
R1
R2
OOH
fatty acid hydroperoxide
O
R1
R2
R1
OH
PUAs
O
fatty acid hydroxide
R1
R2
OH
fatty acid epoxy alcohol
2. Results and Discussion
2.1. Effect of PUAs on Sea Urchin Cleavage and Hatching
Sea urchin eggs have been extensively used to rapidly screen for bioactive compounds interfering
with cell division processes [19] since they can be obtained in large numbers, may be fertilized under
controlled conditions, and have synchronous development. Here we confirm the deleterious effect of
PUAs such as 2-trans,4-trans-decadienal (decadienal), 2-trans,4-trans–octadienal (octadienal),
2-trans,4-trans,7-octatrienal (octatrienal), 2-trans,4-trans-heptadienal (heptadienal) on early and later

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Mar. Drugs 2010, 8
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developmental stages of P. lividus. PUAs blocked sea urchin cell cleavage in a dose dependent
manner, but at different concentrations depending on the chain length of the molecules. Figure 2a
reports the percentage of P. lividus embryos that were blocked after incubation with test compounds
added to seawater at concentrations ranging from 0.658 to 32 μM. Percentage blockage of cell
cleavage increased with increasing chain length from C7 to C10 PUAs, with arrest occurring at
27.27 µM with heptadienal, 16.13 µM with octadienal, 11.47 µM with octatrienal (which was slightly
more active compared to octadienal), and 5.26 μM of decadienal. The saturated aldehyde tridecanal,
also found in diatoms, did not interfere with first cleavage up to 25 µM. Higher concentrations were
not tested due to solubility problems for this compound in sea water.
Figure 2. (a) Cleavage inhibition in sea urchin embryos following PUAs treatment.
(b) Percentage of hatched sea urchin larvae. Sea urchin embryos were treated at increasing
concentrations of 2-trans,4-trans-decadienal (●), aldehyde mix (see text) (●),
2-trans,4-trans-octadienal (♦), 2-trans,4-trans,7-octatrienal (□), 2-trans,4-trans-
heptadienal (▼) and tridecanal (■). Values (means ± S.D.; N = 600) are the results of three
different experiments.