Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae)

Abstract

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR-derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA-exposed S. marinoi cells were treated with the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an opposite response was observed, with an increase in DHR-derived green fluorescence. A higher DHR-derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2-methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O2 (•) generation and SOD activity in H2 O2 formation (with downstream ROS generation dependent from H2 O2 ) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA-exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O2 (•-) and H2 O2 . This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.

The Effect of Polyunsaturated Aldehydes on Skeletonema marinoi (Bacillariophyceae): The Involvement of Reactive Oxygen Species and Nitric Oxide

“…This would also be consistent with the observed concomitant decrease of NO and is supported by the strong effect of the ROS scavenger used in our experiments (Tempol), which is also known to attenuate peroxynitrite [66]. Accordingly, preliminary experiments [48] indicate a major involvement of superoxide anion (O 2 − ) and superoxide dismutase (SOD) activity with consequent H 2 O 2 formation (and H 2 O 2 -dependent downstream ROS generation) in PUA-exposed SM cells. The data were obtained using two SOD inhibitors, 2ME (2-methoxyestradiol) and DETC (sodium diethyldithiocarbamate trihydrate) and suggest an involvement of the H 2 O 2 -dependent oxidation of DHR123, as previously reported [67,68,69].…”

“…In Skeletonema costatum (now replaced as S. tropicum [47]), Chung et al [23] observed NO production in response to light stress, and this was also confirmed in SM [48]. This suggests that NO production in SM is stress-type dependent.…”

Non-Enzymatic Synthesis of Bioactive Isoprostanoids in the Diatom Phaeodactylum following Oxidative Stress

“…and thromboxanes. This phenomenon is not specific to all diatoms, since other species, such as S. marinoi, have oxylipin biosynthetic pathways producing prostanoids (Gerecht et al, 2011;Gallina et al, 2016;Di Dato et al, 2017). A first line of investigation focused on the possible production of polyunsaturated aldehydes (PUAs).…”

“…B3LF83 and Q9FPM6;Feussner and Wasternack, 2002). Therefore, P. tricornutum lacks the enzymatic equipment to produce cyclopentane-oxylipin and differs from other diatom species, such as S. marinoi, in which cyclopentane-oxylipin enzymatic pathways have been described (Gerecht et al, 2011;Gallina et al, 2016;Di Dato et al, 2017).…”

“…Neither could we detect any homolog of enzymes involved in the synthesis of thromboxane (Funk, 2001), which has an ether-containing ring in place of a cyclopentane, or enzymes involved in the synthesis of noncyclic oxylipins in plants (Feussner and Wasternack, 2002). P. tricornutum, therefore, lacks the enzymatic equipment to produce cyclopentaneoxylipin and differs from other diatom species, such as Skeletonema marinoi, in which enzymatic pathways have been described (Gerecht et al, 2011;Gallina et al, 2016;Di Dato et al, 2017). Therefore, the presence of a high basal level of oxidized FAs (5%-7%) reported in the lipidome of P. tricornutum (Collins et al, 2016), with apparently a high structural but unresolved diversity, is puzzling.…”