Biologically Active Oxylipins from Enzymatic and Nonenzymatic Routes in Macroalgae

Marine algae are rich and heterogeneous sources of great chemical diversity, among which oxylipins are a well-recognized class of natural products. Algal oxylipins comprise an assortment of oxygenated, halogenated, and unsaturated functional groups and also several carbocycles, varying in ring size and position in lipid chain. Besides the discovery of structurally diverse oxylipins in macroalgae, research has recently deciphered the role of some of these metabolites in the defense and innate immunity of photosynthetic marine organisms. This review is an attempt to comprehensively cover the available literature on the chemistry, biosynthesis, ecology, and potential bioactivity of oxylipins from marine macroalgae. For a better understanding, enzymatic and nonenzymatic routes were separated; however, both processes often occur concomitantly and may influence each other, even producing structurally related molecules.


Introduction
Fatty acids are key components of cell membranes and storage lipids in all living organisms. These central building blocks are prone to undergoing oxidation reactions through both enzymatic and nonenzymatic cellular mechanisms. The biosynthesis of oxygenated derivatives of polyunsaturated fatty acids (PUFA), collectively termed oxylipins, is highly dynamic and occurs as both a developmentally regulated mode and a response to abiotic and biotic stresses. The oxylipin pathway is initiated by the formation of fatty acid hydroperoxydes, either by chemical (auto)oxidation induced by free radicals and reactive oxygen species (ROS), or catalyzed by enzymes, such as lipoxygenases (LOX) [1]. The primary hydoperoxyde products are further converted into a large variety of oxylipin classes, through an array of alternative and subsequent reactions, having crucial signaling functions in different organisms [2][3][4][5][6][7][8][9][10]. In fact, oxylipins' cellular functions are as diverse as oxylipins themselves [1]. This family of structurally diverse metabolites is ubiquitously distributed in nature, being found in animals, plants, bacteria, mosses, and algae [2,11].
Due to the wealth of novel oxylipin structures encountered in marine organisms, the uniqueness of their biosynthetic pathways, and the potency of their biological effects, marine oxylipins have been recent targets of lipid research. In fact, over the last decades researchers have focused their attention on the isolation, structural elucidation, and biological properties of oxylipins from marine organisms, which have emerged as incredibly rich sources of these low-molecular-weight lipids. The overwhelming majority of marine oxylipins derive from LOX metabolism of PUFA precursors with a variety of carbon lengths (C 16 to C 22 ) and unsaturation patterns (ω3, ω6, ω9) [3].

Rhodophyta
Red algae have provided interesting models to investigate the evolution of the fatty acid metabolism and the function of oxylipins in photosynthetic organisms [63]. Among the different algal phyla, Rhodophyta has been, in fact, the most prolific source of oxylipins, predominantly as result of widespread LOX metabolism, in which C 20 PUFA, namely eicosapentaenoic (4) and arachidonic (6) acids, as well as C 18 PUFA (linoleic (1) and α-linolenic (2) acids), are employed as substrates. The majority of oxylipin structures characterized so far in red macroalgae comes from the metabolism of C 20 PUFA via 12-LOX activity. Nevertheless, other enzymes, including arachidonate 5R-, 8R-, and 15S-LOX, as well as linoleate 9S-and 13S-LOX, were detected in red algae [3,26].
Kumari et al. [24] assessed the content of nine different endogenous hydroxy-oxylipins in forty species belonging to the three algal phyla. Among Rhodophyta, the total oxylipin content ranged from 19.4˘2.2 (Laurencia cruciata Harvey) to 1,753.1˘268.2 ng/g (Gracilaria corticata v. folifera), fresh weight [24]. Despite the large variability observed, which could be attributed to the availability of their substrate fatty acids, species-specific LOX activity, or to other factors, the red macroalgae showed to be particularly rich sources of hydroxyeicosatetraenoic acids [24].
A recent study conducted by Kumari et al. [88] demonstrated the effects of methyljasmonate in the thalli of Gracilaria dura (C. Agardh) J. Agardh. Although the occurrence of methyljasmonate in macroalgae is still not clear, it is widely presumed that, analogously to higher plants, this active form of jasmonic acid regulates a plethora of developmental and stress responses. In fact, methyljasmonate revealed to be a strong elicitor of ROS production in G. dura thalli, leading to the induction of a fatty acid oxidation cascade, which resulted in dose-and time-dependent synthesis and accumulation of several hydroxy-oxylipins, as well as in the upregulation of 13-LOX pathway [88].

Ochrophyta
Ochrophyta has emerged as a source of structurally unique oxylipins derived from novel pathways. As in Rhodophyta, both C 18 and C 20 PUFA are employed as substrates for LOX and other enzymatic systems, such as HPL [2]. The brown macroalgae, particularly those of the order Laminariales, also known as kelps, have been mainly reported to exhibit arachidonate 12-and 15-LOX activities, which catalyze the formation of a number of hydroxylated fatty acid derivatives, short chain aldehydes, and carboxylic oxylipins.
The biogenesis of these metabolites is likely to involve the oxidation of a C 18 PUFA catalyzed by 13-LOX, leading to the formation of a 13-hydroperoxide compound, which subsequently undergoes a number of rearrangements. Moreover, Kousaka et al. [22] evaluated the antibacterial capacity of the isolated oxylipins against two bacterial strains (Bacillus subtilis Cohn and Staphylococcus aureus Rosenbach). The halogenated oxylipins displayed a moderate inhibition against both bacteria [22].  Figure 2d) acids, suggesting 15-LOX activity [18]. In terms of biological activity, 13S-HODE (74) has been shown to induce apoptosis in colorectal cancer cells by down-regulation of peroxisome proliferator-activated receptor (PPAR)-δ [107], also exhibiting remarkable tumor necrosis factor (TNF)-α inhibitory activity (52% and 98% inhibition at 50 µM and 100 µM, respectively) [60]. Additionally, previous studies demonstrated that 15S-HEPE (77) inhibits the growth and the production of arachidonic acid (6)-derived metabolites in human prostatic cancer cells, presumably by PPAR-γ activation [108].
Moreover, three divinyl ether-fatty acids (78-80, Figure 13) were found in L. sinclairii, which are indicative of a LOX with ω6 specificity. Later, the analysis of an extract from L. sinclairii led to the isolation of neohalicholactone (81, Figure 13), a cyclopropyl-containing oxylipin firstly isolated from the marine sponge Halichondria okadai Kadota [105]. Several hydroperoxides deriving from a LOX-catalyzed oxygenation of arachidonic acid (6) were detected in the edible species Laminaria angustata Kjellman [109,110]. These LOX-derived fatty acid hydroperoxides were found to be the intermediate products of C 6 and C 9 aldehyde formation via the action of HPL. Boonprab and co-workers [109] showed that L. angustata produces C 9 aldehydes, namely 3Z-nonenal (82) and 2E-nonenal (60), exclusively from C 20 PUFA, whereas the C 6 aldehyde n-hexanal (83) derives either from C 18 or from C 20 fatty acids (Figure 11b) [109]. Similarly to higher plants, these short-chain aldehydes appear to exert vital functions in chemical attraction and defense [6,110].
A growing body of evidence has been supporting the pivotal role of different oxylipins in defense induction of marine brown algae. Küpper et al. [28] found that bacterial lipopolysaccharides can be strong triggers of early events of defense reactions in the kelp species Laminaria digitata (Hudson) J.V. Lamouroux. It was shown that the challenge of L. digitata sporophytes resulted in an oxidative burst and the rapid release of free saturated and unsaturated fatty acids, with concomitant accumulation of oxylipins, such as 13-hydroxyoctadecatrienoic (13-HOTrE) (84, Figure 2b) and 15-hydroxyeicosapentaenoic (15-HEPE) (85, Figure 2d) acids [28]. The latter was found to inhibit the production of proinflammatory mediators in rat basophil leukemia (RBL)-1 cells [85].
Later, Küpper et al. [30] demonstrated that free PUFA, as well as methyljasmonate, were responsible for triggering oxidative burst in young L. digitata sporophyte thalli, which consequently activated a range of downstream signaling events, including fatty acid oxidation pathways [30]. Further studies evidenced that PGA 2 (44) was able to induce a more powerful oxidative burst than the response triggered by most of the chemical elicitors in L. digitata. However, rather few effects at other levels of signal transduction were observed. PGA 2 (44) did not induce the release of free fatty acids, and only 15-hydroxyeicosatetraenoic acid (15-HETE) (86, Figure 2f) was found to be upregulated in L. digitata [34].
Until 2010, global molecular analyses of brown algal stress response were hampered by the lack of genomic resources. The access to Ectocarpus siliculosus (Dillwyn) Lyngbye genome sequence by Cock et al. [111] represented a major breakthrough in algal research. For instance, although the endogenous occurrence and relevance of jasmonates in macroalgae are still unclear, the presence of AOS and AOC genes involved in the initial step of jasmonates' biosynthesis, in contrast with the absence of genes for jasmonic acid carboxyl methyl transferase in the Ectocarpus genome, suggested that (i) jasmonates may not have the same function in brown algae as in land plants or (ii) they have evolved to serve similar functions using different regulatory systems [42,88]. In fact, the accumulation of C 18 cyclic oxylipins like 12-oxo-phytodienoic acid (12-OPDA) (87, Figure 14), the biosynthetic precursor of jasmonates, as well as of a number of C 20 cyclic prostaglandins was described in L. digitata and E. siliculosus under copper stress, supporting the role of putative cyclopentenones in the defensive mechanisms of brown algae [40,42]. Kumari et al. [24] assessed the content of nine different endogenous hydroxy-oxylipins in seven macroalgae species belonging to Ochrophyta. The total oxylipin contents ranged from 345.4˘56.8 (Scytosiphon lomentaria (Lyngbye) Link) to 2574.5˘155.5 ng/g (Stoechospermum marginatum (C. Agardh) Kützing), fresh weight [24]. The presence of oxylipins in a number of species belonging to Ochrophyta suggests that this group of macroalgae offers a potential source of these biologically active fatty acid derivatives. Years before Rorrer et al. [112] established cell suspension cultures of L. saccharina for the commercial production of hydroxy fatty acids derived from both C 18 and C 20 PUFA [112]. However, the low production of algal biomass, along with their poor ability to utilize exogenously supplied PUFA, rendered oxylipin production a failure on a commercial scale [24]. In more recent years, novel methods for oxylipin production from different PUFA were patented [113][114][115].

Chlorophyta
Unlike Rhodophyta and Ochrophyta, studies on the green algal oxylipin chemistry are much scarcer. As members of Chlorophyta are typically rich in C 18 PUFA, similar trends are expected to reflect for their oxidized derivatives. Besides the action of 9-or 13-LOX already described in green macroalgae, the action of HPL is also characteristic of the oxylipin pathways in this algal phylum, which results in the production of a variety of short-chain carbohydrates, aldehydes, and alcohols [2].
More recently, Kumari et al. [24] determined the content of hydroxy-oxylipins in several species of macroalgae, having found that the ones belonging to Chlorophyta contained the highest amounts of these oxidized metabolites (from 141.2˘12.2 ng/g fresh weight in Codium dwarkense Børgesen to 8161.9˘253 ng/g fresh weight in Chaetomorpha linum (O.F. Müller) Kützing), particularly octadecanoids. Despite the dominance of C 18 PUFA, arachidonic acid (6)-dependent LOX activity was also found, exhibiting 8-, 12-, and 15-LOX isoforms similar to that of arachidonate 11-LOX activity previously reported in U. fasciata, U. conglobata, and to arachidonate 12-, and 15-LOX in E. intestinalis [24]. The genus Ulva has gained worldwide prominence and has emerged as a model for investigating complex metabolic networks, due to its high growth rate and innate ability to grow in wider environmental conditions. In this regard, lipidomic and biochemical changes induced by various stress conditions have been investigated in the species U. lactuca [39,46,47]. For instance, this intertidal alga was able to cope with nitrate and phosphate nutritional stress by altering the metabolic pathways involved in lipid biosynthesis, including a shift in lipid classes, fatty acids, and oxylipins [47]. The alteration of lipid content is known to be one of the most important adaptation strategies to nutrient imbalance in macroalgae. The increased availability of PUFA in nutrient-supplemented U. lactuca thalli led to an increased LOX activity, concomitant with the increase in hydroxy-oxylipin compounds, which have already exhibited defensive roles against oxidative stress conditions in macroalgae [39,46]. The increase in LOX activity and in the relative contents of several hydroxyoctadecadienoic, hydroxyoctadecatrienoic and hydroxyeicosatetraenoic acids suggest the upregulation of different enzyme isoforms, including linoleate 9-LOX, linolenate 13-LOX, as well as arachidonate 5-, 8-, 12-, and 15-LOX [47]. In contrast, the levels of hydroperoxy-oxylipins decreased after nutrient supplementation, pointing to a ROS-mediated nonenzymatic lipid peroxidation due to nutritional limitation-induced oxidative stress.

Nonenzymatically-Derived Algal Oxylipins: The Phytoprostanes
Before it became possible for enzymatic oxylipin signaling pathways to evolve, another reaction sequence that gives rise to a great variety of oxylipins was already present in all aerobic PUFA-containing organisms: free-radical-catalyzed nonenzymatic lipid peroxidation. This early chemical process, which has prevailed throughout the evolution of the oxylipin pathways, can be catalyzed by ROS, which are generated continuously during normal aerobic metabolism [121]. However, a massive production of ROS can likely represent a hallmark of defense responses to a variety of abiotic and biotic stresses. Nonenzymatic reactions are therefore widespread in organisms, even in healthy ones, and because they often evade genetic studies, their relevance can be difficult to estimate. Nonenzymatic lipid oxidation is usually viewed as deleterious; however, recent evidence suggests that during stress, both lipid peroxidation and reactive electrophile species (RES) generation can eventually benefit cells [122].
Phytoprostanes are the resulting products of the autoxidation of α-linolenic acid (2), one of the most abundant PUFA in terrestrial plant membranes, being also present in macroalgae. So far, most studies focused on the nonenzymatically-derived oxylipins from higher terrestrial plants and information regarding the occurrence of this large family of biologically active oxidized lipids in macroalgae is still scarce. However, the presence of α-linolenic acid (2), the known precursor of phytoprostanes, in macroalgae, along with the broad fluctuations of environmental conditions that characterize the marine ecosystem, suggest that macroalgae could be valuable sources of phytoprostanes [123].
The biosynthesis of phytoprostanes ( Figure 15) is proposed to be initiated by the attack of ROS to α-linolenic acid (2), yielding a linolenate radical that readily oxidizes and cyclizes to complex regio-and stereoisomeric prostaglandin-like compounds [124]. Two regioisomeric series (16-and 9-series) can be generated according with the position where the hydrogen abstraction occurs and the oxygen atoms are inserted into the PUFA backbone [125]. G 1 -phytoprostanes (98) can spontaneously decay, forming malondialdehyde (MDA) (99), or be the precursors of different classes of phytoprostanes, named in analogy with the prostaglandin nomenclature system as A 1 (100), B 1 (101), D 1 (102), E 1 (103), F 1 (104), dJ 1 (105), and L 1 (106) phytoprostanes, the latter being the regioisomer of B 1 -phytoprostanes (101) [126]. Thus, a myriad of oxygenated lipids is generated, some of which remain anchored in membranes, while others are released. Ritter et al. [42] have recently described the accumulation of A 1 -phytoprostanes (100) in the brown macroalgae E. siliculosus subjected to copper stress, thus supporting the occurrence of ROS-mediated lipid peroxidation processes [42]. These results also suggest the involvement of phytoprostanes in macroalgae defense responses. In fact, previous reports in land plants have shown that phytoprostanes exert a wide range of biological activities, inducing, for instance, the biosynthesis of secondary metabolites, the expression of genes involved in detoxification processes, and the regulation of the oxidative stress-related mitogen-activated protein kinase (MAPK)-dependent signaling pathway [127][128][129]. Despite these observations, the exact role and physiological function of phytoprostanes have not been yet fully elucidated.
In a recent study conducted by our research group [123] the naturally occurring free phytoprostane composition of 24 macroalgae species was determined, using a fast, selective, and robust ultrahigh-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) method. The analysis of phytoprostanes in natural matrices is extremely challenging, requiring highly sensitive and specific tools for their profiling and characterization [130]. Additionally, the great diversity granted by the presence of racemic mixtures of phytoprostanes increases the complexity of these analyses. The phytoprostane qualitative and quantitative profiles varied greatly among all macroalgae samples (Figure 16), F 1t -phytoprostanes, comprising both 9-F 1t -phytoprostane (107) and 9-epi-9-F 1t -phytoprostane (108), being the dominant class, and L 1 -phytoprostanes (106) the minor one. The brown alga species Cladostephus spongiosus (Hudson) C. Agardh and the green alga Codium tomentosum Stackhouse exhibited higher diversity of compounds, containing 9-F 1t -phytoprostane (107), 9-epi-9-F 1t -phytoprostane (108), 16-B 1 -phytoprostane (109) and L 1 -phytoprostanes (106). The brown alga Bifurcaria bifurcata R. Ross presented the lowest total phytoprostane contents (5.68˘1.09 ng/100 g, dry algae), whereas Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders cultivated in an integrated multitrophic aquaculture (IMTA) system was the richest sample (1,380.90˘103.83 ng/100 g dry algae). However, no conclusion regarding the advantages of IMTA systems could be drawn, as no marine counterpart of this species was analyzed. Moreover, no correlation between the amount of α-linolenic acid (2) in macroalgae material and total phytoprostane content was found, and no phylogenetic relationship was established. Altogether, the collected data suggested that the variations observed in terms of phytoprostane composition could be partially explained by intrinsic factors (e.g., physiological variations within algae organs) and/or extrinsic factors (e.g., geographical origin or area of cultivation, seasonal and environmental variations, time of harvest, water temperature, salinity levels, and processing methods) [123]. Currently, the interest in phytoprostanes targets two general areas: their use as biomarkers of oxidative stress in plant-derived foodstuffs and as bioactive mediators with potential benefits in different biological systems. Evidence points to the involvement of certain phytoprostane classes in the regulation of immune function in humans. E 1 -phytoprostanes (103), previously identified in pollen, inhibited dendritic cell interleukin-12 (IL-12) production and increased T helper type 2 (Th2) cell polarization in vitro [131]. In contrast, Guttermuth et al. [132] found that both E 1 (103) and F 1 (104) phytoprostanes partially inhibited Th1 and Th2 cytokine production in vivo [132]. The immunomodulatory effects of E 1 -phytoprostanes (103) were found to occur via peroxisome proliferator-activated receptor (PPAR)-γ and nuclear factor-κB (NF-κB)-dependent mechanisms [133,134]. Karg and co-workers [135] reported that A 1 (100) and dJ 1 (105) phytoprostanes displayed anti-inflammatory effects in human embryonic kidney (HEK) cells and RAW264.7 murine macrophages, by down-regulating NF-κB and inhibiting nitric oxide (NO) synthesis, respectively [135]. Recently, Minghetti et al. [136] showed that B 1 -phytoprostanes (101) were biologically active in experimental models of immature cells of the central nervous system, exhibiting neuroprotective effects against oxidant injury induced by hydrogen peroxide and promoting myelination through PPAR-γ activation [136].

Conclusions
A large variety of unique oxylipin classes have been found in marine macroalgae, deriving from both developmentally regulated processes (catalyzed by enzymatic systems) and in response to environmental changes (chemical (auto)oxidation). Combined enzymatic and nonenzymatic peroxidation builds the natural peroxide status of membranes. It is the further rearrangement or metabolism of membrane lipid peroxides, by enzymatic and nonenzymatic mechanisms, that results in the accumulation of a far greater variety of secondary oxidation products.
Although oxidized fatty acids are widely distributed in Rhodophyta, Ochrophyta, and Chlorophyta, each major group of algae exhibits its own unique oxylipin signature in terms of fatty acid precursors and typical sites of oxidation. Enzymatically-derived oxylipins from Rhodophyta, most of which result from the metabolism of C 18 and C 20 PUFA, have been the most extensively studied. As in red algae, Ochrophyta species use both C 18 and C 20 PUFA as substrates for LOX (mostly 13-LOX) and other enzymatic systems. Besides the common oxygenated fatty acid derivatives, both red and brown macroalgae have also revealed a high diversity of unusual and unprecedented oxylipin structures. Studies on oxylipin metabolism in Chlorophyta are much scarcer. Existing data point to a dominance of 9-LOX and HPL activity, resulting in the production of several short chain carbohydrates, aldehydes and alcohols. On the other hand, the occurrence and distribution of algal oxylipins from nonenzymatic reactions is highly unpredictable, differing between species and as a consequence of the surrounding growth conditions. Macroalgae thrive in a complex seawater environment, being continuously challenged by an array of potentially pathogenic organisms and multivariate ecological changes. Algal oxylipins may then help to control interactions with other organisms and with the environment, promoting algae survival. Besides the eco-physiological role of these oxidized lipid-derivatives and their relevance in macroalgae, there is still a debate on the exact mechanisms of stress tolerance. Moreover, and because metabolites of this class also play a crucial role in both mammalian physiology and disease, interest in the structural chemistry, biosynthesis, and pharmacological activities of these marine products has increased.
All this evidence supports the need for stronger efforts to improve our knowledge of the pathways of oxylipin biosynthesis, their individual role in cellular responses, and the target elements involved in gene regulation, using the combined "omics" approach of genomics, transcriptomics, and metabolomics.