Saccharina Latissima: Possible Solution for Future Global Protein Shortage?

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MSc Chemistry

Analytical Sciences

Literature Thesis

Saccharina Latissima: Possible Solution for Future

Global Protein Shortage?

The Benefits and Doubts Considering its Application in Industry.

by

Amber A.A. Beerman

12385255 May 2021 12 ECTS September 2020 – May 2021 Supervisor/Examiner: Examiner: dr. A. Astefanei dr. A. Praetorius

Wageningen Food Safety Research

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Abstract

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Over time, seaweed has gained an ever growing interest, due to its environmental friendly nature and nutritious characteristics. The Saccharina latissima (S. latissima), a brown macroalgae that belongs to the Laminariales family, is one of the seaweed species that has great potential to function as a new protein source. However, in addition to its many valuable compounds, the seaweed also contains multiple contaminants, including iodine, arsenic, lead, cadmium and mercury. As of today, the EU has not set any limits with respect to these contaminants for seaweed consumption. This literature study aims to provide new insights into the correlation between nutrient and contaminant uptake and metabolism, in addition to an evaluation of the risks that seaweed consumption might pose to today’s society. The primary nutrients that the S. latissima needs for prosperous growth include phosphate nitrate and carbonate, of which nitrate was proposed as the limiting nutrient. Furthermore, an important revelation includes the relationship between phosphate and arsenic, which likely compete with one another to enter the cell. Therefore, it is likely that a lower phosphate : arsenic ratio increases the probability of arsenic entering the cell. Upon entering the cell, arsenic is primarily biotransformed into arsenic containing lipids and sugars. Therefore, it is difficult to evaluate the safety of seaweed consumption, as each arsenic species differs in bioavailability and cytotoxicity. Arsenic containing lipids were proposed as most bioavailable arsenic species. Furthermore, the cytotoxicity of these arsenic species were comparable to inorganic arsenic. The bioavailability and cytotoxicity of the arsenic containing sugars was almost negligible in vitro. However, these arsenic species were largely bioavailable in vivo, therefore, the possibility exists that these species undergo presystemic metabolization and enter the circulatory system in a different form.

It was proposed that iodine uptake can be divided into three parts: (1) iodide oxidation at the algae surface, catalysed by vanadium-dependent haloperoxidases and Fe(lll), (2) passing the cell membrane through iodination of polyunsaturated fatty acids and (3) reduced back to iodide upon entering the cell. The bioavailability of iodine originating from seaweed was also evaluated. It is proposed that the bioavailability depends on two aspects: (1) composition of the seaweed and how these components behave upon entering the gastrointestinal tract, (2) the form(s) in which iodine exists in the seaweed.

In consequence of these results, future studies are recommended that focus on arsenic and iodine speciation within the S. latissima, as well as mapping the arsenic biotransformation, bioavailability and cytotoxicity throughout the human body. This study should be conducted concurrently to a multivariate study that further establishes the relationships between the nutrients and contaminants, including all parameters related to the growth. It is important to both consider the economic value of the S. latissima, as well as, the safety of S. latissima consumption. Combined, these studies could improve the cultivation environment, as well as, providing a better understanding of the risks that this seaweed poses to today's society.

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Preface

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This literature thesis resembles an important part of my Master’s degree at the UVA/VU. The thesis is the product of a six month literature study that was conducted at Wageningen Food Safety Research, within the business unit Contaminants & Toxins.

I am grateful to all the people that contributed academically, practically and with support to this study. Up and foremost I would like to thank my daily supervisor Siebren van Tuinen for his guidance, support, valuable input and constructive comments throughout this literature project. Furthermore, I would like to thank Karsten Beekmann for his input and feedback during the project. Lastly, I would like to thank everyone within the WFSR seaweed team that provided additional input, knowledge and constructive comments throughout the project.

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List of abbreviations

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ArsI - Arsenic-inducible gene

AsFA - Arsenic containing fatty acid AsHC - Arsenic containing hydrocarbon AsPL - Arsenic containing phospholipid AsSug - Arsenic containing sugar

ATP - Adenosine triphosphate

BBB - Blood brain barrier

Caco-2 - Human epithelial cells

CBB - Calvin Benson Bassham

CCM - Carbon concentrating mechanism

DBL - Diffusion boundary layer

DIC - Dissolved inorganic carbon

DIN - Dissolved inorganic nitrogen

DIP - Dissolved inorganic phosphorus

EC - Effective concentration

EFSA - European Food Safety Authority GERD - Gastroesophageal reflux disease

GIT - Gastrointestinal tract

HepG2 - Human liver cells

IARC - International Agency for Research on Cancer

IC - Inhibitory concentration

LUHMES - Human brain cells

PBCEC - Porcine brain capillary endothelial cells L. saccharina - Laminaria saccharina

NADPH - Nicotinamide adenine dinucleotide phosphate PUFA - Polyunsaturated fatty acid

SFCA - Short chain fatty acid S. latissima - Saccharina latissima

UROtsa - Human urothelium cells

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Introduction

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Over the past decade, seaweed has become an increasing point of focus within Europe, due to its environmental friendly nature and nutritional characteristics. Seaweed is grown within the marine environment and therefore does not need freshwater supply nor does it require any land area [1]. The Saccharina latissima, also referred to as S. latissima, is a brown macroalgae that belongs to the Laminariales family and is more commonly known as sugar kelp. Brown algae represent one of the few eukaryotic species that have developed multicellularity [2]. S. latissima contains many valuable compounds such as essential amino acids, minerals, phenolic compounds, polyunsaturated fatty acids and antioxidants [1]. Within the Netherlands, the S. latissima is cultivated on seaweed farms that are placed at various locations along the coast of the North Sea and at the Eastern Scheldt. A seaweed farm or island consists of multiple seaweed lines that are connected to stakes, an example of such a farm is given in Figure 1.

Nevertheless, these species also contain various contaminants that could pose a potential threat for the consumers. A primary contaminant of interest is iodine, as concentrations up to 14.000 mg/kg have been reported by the WFSR [3]. Furthermore, high levels of heavy metals have also been reported for the S. latissima, which include but are not limited to arsenic, lead, cadmium and mercury, see Table 1. As of today, the EU has not set any limits with respect to these contaminants in seaweed.

This literature thesis aims to provide new insights into the possible correlation between nutrient and contaminant uptake and metabolism. Additionally, the thesis will also discuss the following matters: the need for contaminant speciation and reduction, potential risks that are associated with human seaweed consumption, industrial and biomedical applications, data analysis and future perspectives and challenges.

Figure 1: Example of seaweed farm/island for cultivation of S. latissima [4, 5]

Table 1: Current recommendations versus reported values of frequently found contaminants in S. latissima

Iodine Arsenic (totAs/ iAs) Cadmium Lead Mercury

WFSR recommendations (mg/kg) 20* 40*/2* 3** 3** 0.1**

Reported values (mg/kg) 170-14000 16.7-46.3/0.68-2.1 0.072-0.342 0.37-9.41 0.0222-0.1079

* No formal legislation concerning nutrition

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Chapter 1: Seaweed uptake and metabolism of nutrients

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A primary component of the seaweed physiology includes the nutrients, which comprise of phosphorus, nitrogen and carbon. These nutrients are key components of phospholipids, nucleic acids, adenosine triphosphate (ATP) and are involved in numerous metabolic pathways [6]. These elements are mainly acquired in inorganic forms. Once these nutrients have passed the diffusion boundary layer (DBL) and cell wall, they are transported into the cell via passive diffusion, active transport or facilitated transport. The nutrients are then stored in various pools within cellular vacuoles or are assimilated [7]. This chapter will focus on the uptake and assimilation of the nutrients and discuss relevant pathways. Additional information that was acquired on the various metabolic pathways can be found in Appendix 1, Appendix 2 and Appendix 3.

Dissolved inorganic nitrogen

Dissolved inorganic nitrogen (DIN) is the primary limiting factor in the growth of seaweed. Within the ocean, inorganic nitrogen is available in two forms, nitrate (NO3-) and ammonium

(NH4+). Production based on nitrate is termed new production, as nitrate is externally supplied

either from upwelling or from below the thermocline. Nitrate uptake predominates in the winter, as the surface mixed layer is less separated from the cooler deep water, which are rich in nitrate. The temperature rise that occurs in spring causes the formation of the thermocline, thereby separating the cooler nitrate rich water from the surface mixed layer. Therefore, ammonia uptake, also called recycled production predominates in the summer, as the ammonia is endlessly regenerated within the system by the fish and invertebrates that are associated with the seaweeds [7, 8].

Recent studies have shown that the S. latissima requires high ambient nitrate concentrations to maintain rapid growth [9]. Furthermore, the species is not able to compete for the DIN during late spring and summer as the phytoplankton are more efficient [9]. Therefore, the S.

latissima needs to take up most of the nitrate early in the season when the ambient nitrate

concentrations are the highest [9]. During spring and summer the sporophyte growth of the species is mainly based on internal organic and inorganic nitrogen components, such as proteins, pigments and inorganic nitrate [9].

Uptake mechanism and assimilation

Nitrate is taken into the cell via active transport, while ammonia is taken up via passive diffusion [7]. Within the cell, nitrate is either stored in an inorganic pool within cellular vacuoles or assimilated into ammonia, see Figure 2. For the assimilation into ammonia two enzymes are used, nitrate reductase (NR) and nitrite reductase (NiR) [7]. NR converts nitrate into nitrogen dioxide (NO2-), whereafter NiR converts nitrogen dioxide into ammonia [7]. In

contrast to nitrate, ammonia storage is rather limited, as it is rapidly converted into amino acids [7].

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Figure 2. Uptake and assimilation dissolved inorganic nitrogen. Assimilation enzymes: NiR, nitrite reductase; NR, nitrate reductase [7].

Dissolved inorganic phosphorus

Dissolved inorganic phosphorus (DIP) is the second most limiting factor in the growth of seaweed [7]. The phosphorous levels in aquatic ecosystems follow a seasonal pattern, where inorganic phosphorous is released from the sediment in spring, due to the increasing water temperature. The increase in phosphorous levels causes the algae to reproduce in a rapid manner. Primary productivity then declines when most of the phosphorous is converted into organic phosphorous [8].

Uptake mechanism

As of today, very little is known about the DIP uptake in macroalgae [6]. Nevertheless, it is likely that the DIP uptake of macroalgae is similar to the DIP uptake of higher plants, which acquire inorganic phosphorus through phosphate (PO43-). In the proposed mechanism

phosphate is transported into the cell via diffusion, where it can either be stored in an inorganic pool within cellular vacuoles or be utilized in the numerous pathways which require phosphate [6, 7]. Another described mechanism is the utilization of organic phosphorous. Through the external enzyme alkaline phosphatase seaweeds can break down organic phosphorous into phosphate [7]. However, alkaline phosphatase activity in the S. latissima has not yet been studied.

Dissolved inorganic carbon

Dissolved inorganic carbon (DIC) is a crucial nutrient for marine algae and plants in general, due to its vital role in the photosynthesis. There are two forms of DIC, carbon dioxide (CO2)

and bicarbonate (HCO3-). The predominant DIC species in seawater is bicarbonate, as dissolved

carbon dioxide gas is only present in low quantities [10]. Contrary to the majority of the macroalgae, the Laminaria saccharina (L. saccharina), genus of the S. latissima, lacks internal carbonic anhydrase (CA) activity, which catalyzes the dehydration of bicarbonate to carbon dioxide inside the cell. Opposed to internal CA activity, the L. saccharina exhibits external CA activity at the cell membrane. It was therefore suggested that bicarbonate can be dehydrated by the simultaneous operation of two components, (1) proton pumps which create acid zones in the cell wall region near the cell membrane and (2) external CA activity which catalyzes the dehydration of bicarbonate to carbon dioxide. Concurrently, direct uptake of carbon dioxide occurs via passive diffusion [11].

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Uptake mechanism and assimilation

Due to the lack of internal CA activity, it was proposed that the carbon dioxide concentrating mechanism (CCM) is located at the cell membrane [10, 11, 12]. The CCM increases the carbon dioxide concentration to improve the photosynthetic productivity in algal cells. It was proposed that the proton-gradient-driven carbon dioxide pump in the cell membrane is actually integrated with the external CA catalyzed bicarbonate dehydration [10, 11, 12]. The proposed mechanism includes five stages, see Figure 3.

(1) Dissolved HCO3- and CO2 pass the cell wall.

(2) HCO3- is dehydrated into CO2 in the acid region. This reaction is catalyzed by external

CA, while the uncatalyzed dehydration is rather slow. The excess H+_{is excreted from}

the acid region into the bulk medium (seawater).

(3) CO2 passively diffuses into the cell, while direct uptake of HCO3- only occurs at high pH,

most likely as a survival mechanism. The excess H+_{is excreted from the cell via the H}+

-ATPase to support the dehydration of HCO3- in the acid region.

(4) CO2 is transported into the chloroplast.

(5) CO2 is transported into the stroma where it will enter the Calvin cycle.

Figure 3. Uptake and assimilation dissolved inorganic carbon. Assimilation enzymes: CA ext, external carbonic anhydrase.

Photosynthetic and respiratory pathway

The photosynthetic apparatus, which includes light-absorbing pigments, enzymes and electron-carrying chemicals is located within the chloroplast. Photosynthesis can be divided into two stages, (1) the light harvesting reactions which occur in the grana and (2) the carbon-assimilating reactions which occur in the stroma [8]. The light harvesting reactions include two interdependent systems, photosystem 1 and photosystem 2, see Figure 4. The ATP and nicotinamide adenine dinucleotide phosphate (NADPH) that are formed in this pathway are then transported to the carbon assimilating reactions [13].

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Figure 4. Light harvesting reactions [8].

The primary pathway for carbon assimilation in C3 plants, which includes amongst others the

S. latissima, is the Calvin Benson Bassham cycle (CBB cycle). The Calvin cycle, described in

Figure 5, is an anabolic process which includes 13 reactions that are catalyzed by 11 enzymes [8]. These 13 reactions can be divided into three phases, carboxylation, reduction and regeneration [14]. During one cycle two glyceraldehyde-3-phosphate (G3P) molecules are formed, every three cycles one G3P molecule is exported out of the Calvin cycle, either into the chloroplast where it is converted into starch and stored for later use or exported into the cytosol/cytoplasm where it will contribute to the formation of other compounds in the glycolytic pathway or the pentose phosphate pathway. The other five G3P molecules remain in the cycle to regenerate ribulose biphosphate (RuBP), enabling the system to prepare for more CO2 to be fixed [14]. For additional information on the photosynthetic pathway, see Appendix 4.

Figure 5 Fixation of CO2 through CBB cycle. Cycle products: RuBP, ribulose biphosphate; 3-PG, 3-phosphoglycerate; BPG, 1,3-biphosphoglycerate; G3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; FBP, fructose-biphosphate; F6P, fructose-6-phosphate; E4P, erythrose-4-fructose-6-phosphate; SBP, sedoheptulose bifructose-6-phosphate; S7P, sedoheptulose-7-fructose-6-phosphate; R5P, ribose-5-fructose-6-phosphate; Ru5P, ribulose-5-phosphate. Cycle enzymes: RUBISCO, ribulose biphosphate carboxylase; PGK, phosphoglycerate kinase; G3PDH, glyceraldehy de-3-phosphate dehydrogenase; TPI, triosephosphate isomerase; FBA, fructose-biphosphate aldolase; FBPase, fructose biphosphatase; TKL, transketolase; SBPase, sedoheptulose-1,7-biphosphatase; R5Pise, ribose-5-phosphate isomerase; R3epi, ribulose-5-phosphate epimerase; PRK, phosphoribulokinase [14].

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Organisms that make use of photosynthesis also encounter another process which is called photorespiration. Photorespiration is a competing reaction which inhibits photosynthesis. This process includes the uptake of oxygen and the release of carbon dioxide and is caused by a fundamental inefficiency of RuBisCO, as it essentially wastes energy in the form of ATP. The reaction starts in the chloroplast and can be described as the light-dependent uptake of O2 [15, 16, 17]. The pathway consists of 9 reactions that are catalyzed by 9 enzymes directly and 3 enzymes indirectly, see Figure 6 [15, 16, 17]. For additional information on the photorespiratory pathway, see Appendix 5.

The discussed metabolic pathways already highlight the complexity of the processes that are related to the growth of seaweed, specifically since all metabolic processes are somehow related to each other. The primary component in the metabolic processes is G3P. This component is crucial for the production of sugars, which is the primary marketing point of the

S. latissima. Therefore, it is essential to understand which factors contribute to the extent in

which the seaweed is able to grow. Especially since a higher yield will increase the profit for the cultivating company. A primary aspect of this matter is the uptake of nutrients and the extent to which these processes are influenced by external factors, which will be discussed in chapter 5. Additionally, the growth could also be related to the uptake of contaminants, which will be discussed in the following chapter.

Figure 6. Fixation CO2 through photorespiration. Cycle products: RuBP, ribulose biphosphate; 2-PG, 2-phospho-glycolate; glycolate; glyoxylate; glycine; THF-C1, hydroxymethyl transferase-C1; serine; hydroxy-pyruvate; glycerate; PG, 3-phosphoglycerate. Cycle enzymes: RuBisCO, ribulose biphosphate carboxylase; PGP, phosphoglycolate phosphatase; GOX, glycolate oxidase; GGT, glu:glyoxylate aminotransferase; GDC, gly decarboxylase; SHMT, ser hydroxymethyl transferase; SGT, ser:glyoxylate aminotransferase; HPR, hydroxypyruvate reductase; GLYK, glyxerate kinase; CAT, catalase; GS/GOGAT, gln synthase / glu synthase glu:oxoglutarate aminotransferase; mMDH/cMDH/pMDH, mitochondrial/chloroplastic/perixome malate dehydrogenase [15, 16, 17].

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Chapter 2: Seaweed uptake and metabolism of contaminants

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The S. latissima contains many valuable compounds, such as essential amino acids, minerals, phenolic compounds, polyunsaturated fatty acids and antioxidants, making the seaweed attractive for industry. However, the S. latissima also contains high levels of contaminants, including iodine, arsenic, lead, cadmium and mercury, which should be carefully studied before implementing the seaweed in the (intended) industrial products.

To ensure the safety of seaweed consumption, it is important to study and evaluate four aspects: (1) the underlying mechanisms through which the contaminants are able to enter the seaweed cell, (2) the biotransformation of the contaminants upon entering the seaweed cell, (3) the parts in which the contaminants are stored and (4) methods to reduce the contaminant levels. The following chapter will primarily focus on iodine and arsenic, extensively discussing their uptake, storage, metabolism, speciation and potential reduction methods. Lead, cadmium and mercury will only briefly be discussed, mainly due to the lack of information and knowledge that currently exists with respect to these contaminants.

Arsenic

One of the most concerning contaminants present in seaweed is arsenic. Arsenic is a naturally occurring element that is globally present in large quantities that are released into the environment from natural and anthropogenic sources [18]. As of yet, more than 100 arsenic species have been identified in the marine environment. The specific toxicity depends on the molecular nature, ranging from non-toxic to arsenic species that are classified as Class 1 carcinogenic to humans by the IARC [19]. The arsenic concentration in the ocean is generally between 1 and 3 g/L (1 – 4 *10-2_{M), which predominantly constitutes of the inorganic}

forms arsenious acid (iAs(lll)) and arsenic acid (iAs(V)) [20]. Seaweeds and especially brown macroalgae are known to accumulate heavy metals, therefore, their total arsenic levels can be 1.000 to 50.000 times higher than the surrounding ocean levels [21]. An overview of the most important arsenic species and metabolites is given in Figure 7.

Arsenic uptake mechanism

The chemical similarities between arsenic and its fellow group 15 member phosphorus forms an essential part in the understanding of arsenic uptake in algae. One of the predominant arsenic forms within the ocean is iAs(V), in which it exists as the deprotonated oxo-anion H2AsO4-. This arsenic form has a striking resemblance in terms of pKa and ionic radius to

phosphate, which exists in the marine environment as the deprotonated oxo-anion H2PO4-.

The membrane transport systems of the algae are unable to differentiate between these two species, resulting in an easy access for arsenic into the algal cell [22]. Nevertheless, the arsenic is not accumulated in this form, but rather assimilated by processes of methylation and alkylation [23]. The resulting end products, which mainly consist of arsenic containing sugars and arsenic containing lipids are then accumulated within the cells [23]. It is currently believed that low phosphate levels in the ocean contribute to the degree in which iAs(V) is taken up by the cells, which will further be discussed in chapter 5 [24].

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As(lll) MA(lll) DMA(lll)

As(V) MA(V) DMA(V)

AsSug AsB TMA

AsHC AsFA AsPL

Figure 7: Molecular structures of the most important arsenic species in the marine environment. iAs(lll), arsenious acid; MA(lll), monomethylarsonous acid; DMA(lll), dimethylarsinous acid; iAs(V), arsenic acid; MA(V), monomethyl arsonic acid; DMA(V), dimethyl arsinic acid; AsSug, arsenosugar; AsB, arsenobetaine; TMA, trimethylarsine; AsHC, arsenohydrocarbon; AsFA, arsenofatty acid; AsPL, arsenophospholipid.

Arsenic metabolism

Over time, algae have developed a process in which they reduce the potential iAs(V) toxicity, in this process iAs(V) is converted into arsenic containing sugars. One of the most recent proposed metabolic pathways for the assimilation of iAs(V) is given in Figure 8. The first step of the iAs(V) biotransformation includes the reduction to iAs(lll). For the reduction of iAs(V) to iAs(lll) two possibilities exist, either the reaction is facilitated by iAs(V) reductase or by glutathione [25, 26]. The reduction step has already been proven for the brown alga Fucus

serratus (F. serratus) [38].

The following step is the methylation of iAs(lll), the methylation takes place in the presence of S-adenosylmethionine (SAM) and is catalyzed by iAs(lll) methyltransferases and glutathione. As of yet, no consensus has been reached on whether the arsenic methylation can be regarded as a detoxification mechanism, as dimethylarsinous acid (DMA(lll)) and monomethylarsonous acid (MA(lll)) have been proven to be more cytotoxic, genotoxic and reactive than iAs(lll) [27, 28]. The methylation processes can also be reversed by demethylation. However, this has only been studied in microorganisms and microalgae. This process is catalyzed by an arsenic-inducible gene (ArsI), this protein exhibits C-As lyase activity and is therefore able to cleave the C-As bond [29].

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DMA was proposed as a possible precursor for the formation of arsenic containing sugars. It was suggested that dimethyl arsinic acid (DMA(V)) is first reduced to DMA(lll), whereafter the adenosyl group of SAM is transferred to DMA(lll), forming the intermediate dimethylarsinyladenosine. Dimethylarsinyladenosine then undergoes glycosidation with available algal metabolites to form arsenosugars [30, 31]. However, it remains unknown which enzymes are involved in the adenosylation and glycosidation, moreover, evidence to support that the adenosyl group stems from SAM is also lacking. Another possibility that has been proposed, is that arsenic containing sugars are derived from the degradation of arsenic containing lipids [32, 33].

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It was proposed that arsenic containing phospholipids (AsPLs) are formed from arsenic containing sugars [35]. Dimethylarsinoylpropionic acid was suggested as the precursor for the formation of arsenic containing fatty acids (AsFAs) and for the arsenic containing hydrocarbons (AsHCs) it was proposed that they are formed from reduction of free fatty acids, whereafter it is combined with DMA [36, 37]. As of today, all above mentioned processes and enzymes have not yet been confirmed for the S. latissima. Nevertheless, the pathway has partially been proven for the fellow brown alga F. serratus. Results indicated that at low arsenic exposure (20 g/L), iAs(V) was taken up and converted to arsenic containing sugars without significant accumulation of any intermediates. Nonetheless, an exposure level of 50 g/L iAs(V) resulted in an accumulation of monomethyl arsonic acid (MA(V)) and DMA(V). Furthermore, the highest arsenic exposure level (100 g/L) resulted in an accumulation of iAs(lll) and proved to be fatal to the alga. It is likely that this level of arsenic resulted in an overload of the pathway [38].

Arsenic localization

Recently, a study focused on the localization of arsenic in the S. latissima [39]. Results indicated that most of the arsenic was stored in the young frond and sori, whereas only a small amount of arsenic was stored in the stipe and/or midrib with respect to the remaining parts of the seaweed, see Figure 9. The arsenic containing sugars were the most abundant arsenic species. Additionally, a relationship between the AsPLs and the metabolic activity was proposed, since the younger parts contained higher levels of AsPLs than the older parts [39]. Based on these results it was suggested that the AsPLs are stored in the metabolically active thallus parts on purpose or that the AsPLs are produced as a side product [39]. Furthermore, it was proposed that the AsPLs are potentially broken down to arsenic containing sugars or that the AsPLs are formed from arsenic containing sugars that undergo an AsPLs production cycle [39].

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Arsenic speciation

Arsenic studies in seaweed have primarily focused on the inorganic arsenic content with respect to the total arsenic content. Nonetheless, recent studies also started to focus on the speciation of the organic fraction [40, 42]. Within the S. latissima, the arsenic content primarily consists of organic arsenic (95%) [40, 42]. Speciation of the organic fraction is therefore vital to accurately evaluate potential risks, especially since the toxicity varies between the different arsenic species. Moreover, arsenic speciation and localization is also of great importance to determine which processing steps are needed to effectively reduce the arsenic content. In Figure 10 an estimation is given of the arsenic speciation in the S. latissima and in Table 2 a comparison is given of the total and inorganic arsenic content reported by WFSR versus found literature values.

The arsenic levels in Table 2 indicate that the inorganic arsenic content is rather low with respect to the total arsenic content. A proposed explanation for this matter is that iAs(V) is transformed to organic arsenic as effectively as possible, as iAs(V) is able to inhibit the photophosphorylation and oxidative phosphorylation processes, resulting in protein denaturation and cell death. Similarly to inorganic arsenic, the methylated arsenic species are likewise instantly transformed to other organic arsenic species. The contribution of these arsenic species in the S. latissima are therefore almost negligible, see Figure 10.

Figure 10: Arsenic speciation S. latissima. A: Approximate distribution lipophilic, water-soluble and residue fraction B: Arsenic speciation lipophilic fraction, AsFAs excluded due to low fraction (0.05%). C: Arsenic speciation water-soluble fraction. [39, 40].

Table 2: Reported values WFSR versus reported values literature of arsenic content S. latissima

Total arsenic Inorganic arsenic Lit. Reported values WFSR (mg/kg) 16.7 – 46.3 0.68 – 2.10 [3] Reported values literature (mg/kg) 23.3 – 99.1 0.16 – 0.23 [101, 102]

Water-soluble fraction Lipophilic fraction Residue fraction AsSugOH AsSugPO4 AsSugSO3 AsSugSO4 DMA iAs AsHC AsPL A B C

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The data from Table 2 also shows variations in the arsenic levels reported by WFSR with respect to the reported values in literature. It is very complex to attribute these differences to only one factor, since it is presumably induced by a combination of multiple components. The biggest difference is the location in which the S. latissima was cultivated. The reported values from WFSR stemmed from S. latissima that was cultivated near the Dutch coast, while the reported values from literature stemmed from cultivation sites located in Norway, Iceland and France. These spatial variations means that the seaweed was subjected to different environments with variations in the UV-index, temperature, salinity and many more. Additional important components that should be considered are the phosphate and arsenic levels in the ocean. The PO4-:As(V) ratio is dependent on the location, therefore, the degree

in which As(V) competes with PO4- to enter the cell will also depend on the location and will

thereby result in spatial variations in the accumulated arsenic levels. It is viable that these factors combined could have resulted in the observed variations.

Approximately 80% of the total arsenic content constitutes of arsenic containing sugars, these arsenic species are therefore the primary arsenic form in the S. latissima, see Figure 10. As of today, over 15 different molecular forms are known [37]. Generally, arsenic containing sugars constitute of a dimethylarsinoyl moiety. In this moiety, arsenic is pentavalent and bound to two methyl groups, the sugar and oxygen. The various molecular forms only differ in the side chain at the first position of the sugar backbone. The side chains that are primarily found in the S. latissima include glycerol (AsSugOH), phosphate (AsSugPO4), sulphonate (AsSugSO3)

and sulphate (AsSugSO4), see Figure 11. A variant of the arsenic containing sugars include the

thiolated-arsenic containing sugars (thio-AsSug), in which the oxygen is replaced by sulfur [41, 42]. A second variant on the typical arsenic containing sugars are the trimethylarsonium compounds, which are structurally similar to arsenobetaine [43].

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The lipid arsenic fraction in the S. latissima is generally around 10%, see Figure 10. The lipid fraction can roughly be divided into 70 – 72% AsPLs, 28% AsHCs and 0.05% AsFAs [40, 42]. Arsenic containing lipids are organic arsenic species that were first discovered in the marine environment in 1968 and first identified in 1988 [44, 45]. The formation of these arsenic species were investigated through radiolabeling. Radio-chromatograms revealed that the first intermediates in the biotransformation of iAs(V) were arsenic containing lipids. Two of the arsenic containing lipids were converted into water-soluble arsenic compounds, which were later thought to be arsenic containing sugars [46, 47]. Similarly to the arsenic containing sugars, arsenic containing lipids also constitute of a dimethylarsinoyl moiety. Over the years, two AsFAs, eight AsHCs and fourteen AsPLs have been identified in the S. latissima, an overview of these arsenolipids is given in Appendix 4. Results also indicated that the fatty acid groups of the AsPLs were attached to the terminal glycerol moiety and that the hydroxyl groups of the glycerol linker and ribose were not modified [39, 40].

Even though the actual mechanism of the AsPLs formation is currently unknown, all discussed studies allude to a relation between the formation of AsPLs and arsenic containing sugars. A reasonable theory would be that the AsPLs are formed from AsSugPO4. Additionally, it is likely

that the reaction goes both ways and operates at a certain equilibrium. This theory also aligns with the proposed arsenic biotransformation displayed in Figure 8.

Arsenic reduction

Thus far, only a limited amount of studies have focused on the arsenic reduction in seaweed, none of which included the S. latissima. Nevertheless, arsenic reduction studies have been conducted for other seaweed species. For the brown seaweed Hizikia fusiforme (Hijiki) it was reported that heating in water at 90 C for 5 minutes reduced the total arsenic content up to 75% and the As(V) content up to 80%. The Hijiki samples were also treated with NaCl, the highest arsenic reduction was obtained through soaking the samples for 20 min in an 2% NaCl solution, resulting in an total arsenic reduction up to 55% and an As(V) reduction up to 53%. Additionally, using both methods consecutively improved the arsenic reduction up to 15% [48]. Another study found that repeatedly boiling the Hijiki samples reduced the total arsenic content up to 92% [49].

What should be noted is that these studies mainly focused on the reduction of the water soluble arsenic species. Therefore, it is unlikely that arsenic species such as arsenic containing lipids would also be removed. In the following chapter, the potential cytotoxicity of arsenic containing lipids will be discussed. It is therefore recommended, to not only study methods that focus on the total arsenic content, but also focus on the specific reduction of the arsenic containing lipids.

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Iodine

In addition to arsenic, iodine is also a primary concern regarding the safety of S. latissima consumption, as high levels are often found in this species. For example, within European sugar kelps, iodine contents as high as 6500 mg/kg DW have been found [50]. This means that the tolerable Upper Intake Level of 600 g/day, as advised by the European Food Safety Authorities (EFSA), could be exceeded from consumption as low as 92 mg sugar kelp per day, while the average daily consumption was estimated at 3.3 g/day [51, 52].

Iodine exists in the ocean with an approximate concentration of 0.5 M (63 g/L) [53]. Within the ocean, iodine primarily exists in the oxidation forms iodate (IO3-) and iodide (I-), in addition

to smaller concentrations of molecular iodine (I2), hypoiodous acid (HIO) and iodinated

organic compounds [57]. The existence of iodine in seaweed has been known for over 200 years, ever since Bernard Courtois discovered the element by accident in 1811, as he was searching for explosive materials in the ashes of kelp during the Napoleonic Wars [54]. The Laminariales family, which includes the S. latissima, are the strongest iodine accumulators, since this family is able to accumulate iodine within their tissues up to 30.000 times with respect to the surrounding iodine levels [55, 56]. To this day the Laminariales family remain a major source for the recovery of this element [57].

Iodine uptake mechanism

Even though the actual uptake mechanism of iodine remains unknown, various mechanisms have been proposed over the years. Very early on it was already suggested that the enzyme group vanadium-dependent haloperoxidases (VPO) could be involved in the iodine accumulation of brown algae [58]. This enzyme group catalyzes the oxidation of iodide at the algae surface by using hydrogen peroxide as a precursor, forming I2 or HIO [59]. It is suggested

that these oxidized forms, which are more lipophilic than iodide, pass the cell membrane [58]. VPOs have proven to be key enzymes for the halogen metabolism of brown algae [59]. The presence of vanadium-dependent bromoperoxidases in the Laminaria saccharina (L.

saccharina), genus of the S. latissima, was confirmed in 1986 [60]. This VPO group catalyzes

both the oxidation of iodide and bromide and are more commonly known as bromoperoxidases. Within the L. saccharina, the enzyme activity of this VPO group was estimated at 60±20 U/g in the blade and 110±10 U/g in the stipe. The bromoperoxidase activity was proven in both the surface and the cortical-cell protoplasts [61].

The oxidized iodine forms comprise of a rather reactive nature, in consideration of these characteristics, a second proposed theory implied iodine uptake through iodination of the polyunsaturated fatty acids (PUFAs) of the cell membrane. Once it passes the cell membrane, it is presumed that the species are reduced back to iodide through unknown cellular reducing agents. The third and last proposed mechanism implied the Fe(lll) catalyzed oxidation of iodide to molecular iodine, hereafter the oxidized form passes the cell membrane and is likely reduced back to iodide [58]. Recent studies also indicated that iodate could be actively taken up by brown algae, however, the uptake was rather slow and only observable after 24h [62].

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As of yet, no consensus has been reached on the actual uptake mechanism of iodine. Nevertheless, it is likely that the mechanism is a combination of all proposed theories. A feasible proposition would be that iodide is oxidized at the algae surface, catalyzed by both VPO and Fe(lll). Hereafter, the oxidized species pass the cell membrane through iodination of the PUFAs and are reduced back to iodide after entering the cell. It is probable that the capacity in which VPO and Fe(lll) contribute to the oxidation is dependent on various factors such as location, external iron levels and hydrogen peroxide production, of which the latter has been related to stress through changes in the environmental conditions [63].

Iodine storage

For the Laminariales family, it is believed that iodine is stored in non-specialized vacuoles in the blade cortical cells and in phenol-containing physodes in the stipe cells, which are structural components of the cell wall [64, 65]. Iodine is mainly stored in its inorganic form (80-90%), the remainder is stored as iodinated amino-acids, e.g. mono- and di-iodotyrosine [58]. In Figure 12 an overview is given of the most important iodine species. For the L.

saccharina, it is suggested that iodine is translocated towards the meristematic zone, which is

the zone where plant cells undergo fast mitotic division, thereby creating new cells for root growth [64].

Iodide Molecular iodine

Hypoiodous acid Iodate

Mono-iodotyrosine Di-iodotyrosine

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Recently, a study compared the total iodine concentration of the different parts of the S.

latissima, see Figure 9. These results revealed an increase in the iodine concentration from

the blade towards the holdfast [66]. The observed total iodine concentrations included 3341 mg/kg for the blade, 3579 mg/kg for the meristem (located at the bottom of the young frond), 5149 mg/kg for the stipe and 6130 mg/kg for the holdfast [66]. Recent preliminary data in another study suggested that the iodine is located internally in vacuoles of the meristoderm cells [69]. Nevertheless, it remains unknown how iodide is held and/or fixed after it is internalized.

Iodine efflux

Desiccation, high levels of light, atmospheric ozone and reactive oxygen species can induce severe oxidative stress for algae. Over the years, it has been proposed that the stored iodine can function as an inorganic antioxidant in response to the induced oxidative stress [67, 68]. One of its antioxidant mechanisms is the detoxification of atmospheric ozone, in this process atmospheric ozone reacts with the iodide at the algal surface, resulting in the formation of iodine oxides [69, 70]. A second antioxidant mechanism includes the iodine efflux, which enables scavenging of hydrogen peroxide, hypoiodous acid and other reactive oxygen species that are catalyzed by VPO [69].

Recent studies have proposed that the iodide transport across the membrane is facilitated by the presence of molecular iodine. The molecular iodine, which possibly acts as a carrier, is formed via the VPO catalyzed reactions [71]. It was also proposed that the antioxidant function of iodine could explain the depth-dependent variations in the iodine levels. It was suggested that at greater depth, the algal cells are less exposed to the oxidative stress, resulting in lower iodine content [72].

Iodine speciation and variation

As of yet, no studies have focused on iodine speciation in the S. latissima. Nevertheless, iodine speciation studies have been conducted for other seaweeds. For the green seaweed Undaria

pinnatifida (Wakame), it was reported that iodine was both associated with low and high

molecular weight (MW) fractions and mainly constituted of iodide, iodate and the iodoamino acids mono- and di-iodotyrosine. The same study also focused on the brown seaweed,

Laminaria japonica (Kombu), for this seaweed only the presence of iodide was proven [73].

However, another study revealed that the Laminaria japonica contained 88.3% iodide, 10.3% organic iodine and 1.4% iodate [74]. Future iodine speciation studies for the S. latissima are recommended, as the human bioavailability could differ per iodine species, which will be discussed in the following chapter.

Current studies have only reported the total iodine content in the S. latissima. In Table 3 a comparison is given of the total iodine content reported by the WFSR versus reported literature values. The large variation with regards to the total iodine content underlines an important issue and highlights the current knowledge gap. These differences could not be attributed to time nor location, since the WFSR has reported large variations within the same seaweed farm at the same moment in time. It is therefore recommended to conduct additional studies that focus on the relationship between the iodine content and environmental factors such as UV-index, temperature and the external iodine levels.

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Table 3: Reported values WFSR versus reported values literature of total iodine content S. latissima Total iodine content Lit.

Reported values WFSR (mg/kg) 170 - 14400 [3] Reported values literature (mg/kg) 380-7208 [75,76]

Iodine Reduction

Over the years, various methods have been studied that could potentially reduce the iodine content within the S. latissima, including water soaking, boiling, water blanching and freeze-thawing. Water soaking at 32 C for 1-6 hours reduced the iodine content by 84-88% [50]. It is likely that the removed iodine mainly constituted of iodide, as the negative charge greatly increases its water solubility. Water boiling for 2 and 20 minutes resulted in an iodine reduction of 33% and 75% respectively [77]. Water blanching resulted in a reduction up to 88%, however, a nutritionally valuable compound loss of up to 54% was also observed, which mainly included ash. Moreover, it also caused a significant loss of glutamic acid and alanine, which resulted in a higher essential amino acid/amino acid (EEA/AA) ratio. This study also concluded that freeze-thawing did not reduce the iodine content [78].

Other heavy metals

Generally, macroalgae are able to accumulate trace metals as much as a thousand times higher than the surrounding concentrations in the seawater [79]. Within these macroalgae, brown algae have been reported as the highest metal accumulators. These species are unable to regulate the uptake of trace elements, due to the large number of anionic groups in the cell wall. In brown algae, the cell walls mainly consist of sulfated polysaccharides and alginates, which consists of polygalacturonic acid units that contain anionic carboxylic binding sites that are able to interact with metallic cations, these binding sites are displayed in Figure 13 [80, 81, 82].

Generally, the internal concentration is proportional to the bioavailability of the metals in the ocean, except for highly polluted environments. The capacity in which macroalgae can accumulate these metals depends on a variety of factors such as the affinity for each element, metabolic processes, age of the plant, season, nitrogen availability, pH, light, salinity, temperature, wave exposure and location. Nevertheless, the elemental levels are mainly affected by the uptake capacity of the algae and the bioavailability of the metals in the surrounding ocean water [83, 84, 85]. In Table 4 a comparison is given of the heavy metal content reported by the WFSR versus found literature values.

Table 4: Reported values WFSR versus reported values literature of heavy metal content S. latissima

Cadmium Lead Mercury Lit. Reported values WFSR (mg/kg) 0.072 – 0.342 0.37 – 9.41 0.0222 – 0.1079 [3] Reported values literature (mg/kg) 0.2 – 2.0 0.03 – 4.5 0.0009 – 0.1054 [86, 87]

Figure 13: Molecular structure polygalacturonic acid units that are located in the cell wall of brown algae. Figure retrieved from Futo et al. 2003 [88].

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Cadmium exists in the marine environment with an approximate concentration of 0.5 nM (56 ng/L) [89]. Within the marine environment, cadmium can form complexes with chloride and sulfate salts and is primarily found as Cd2+_{, CdCl}+_{, CdCl}

2, CdCl3- and CdCl42-. Cd2+ is the

bioavailable form of cadmium in the marine environment and is considered to be very toxic to both plants and animals [90]. The uptake mechanism is thought to be related to chelation, as studies have shown that cadmium is able to form complexes with the carboxyl groups of the alginate components of the cell wall [91].

Lead exists in the ocean with an approximate concentration of 0.6 pM (0.1 ng/L) [89]. The dissolved organic lead species PbCO3 and Pb(CO3)22- can be found within the ocean. The

uptake mechanism of lead consists of a combination of chelation, ion exchange and reduction reactions, in addition to metallic lead precipitation on the cell wall [91]. Mercury is present in the ocean with an average concentration of 1.5 pM (0.3 ng/L) [92]. Within the marine environment, mercury exists in its elemental form, divalent form, in the organic forms methylmercury and dimethylmercury, additionally it can also form complexes with halides such as chlorine (HgCl3− and HgCl42−) [93, 94]. The uptake mechanism of mercury is primarily

related to the carboxylate groups in the biomass [95].

Uptake mechanism

The uptake of these heavy metals can be divided into two steps [96, 97].

• Step one includes the rapid initial surface reaction in which the metals are adsorbed onto the algal surfaces by electrostatic attraction to the negative sites. This step is independent of the factors that influence the metabolism and mainly depends on the bioavailability of the elements in the surrounding seawater.

• Step two includes the much slower active uptake in which metal ions are transported through the cell membrane into the cytoplasm. This step is mainly dependent on metabolic processes and therefore sensitive to variations such as temperature, light and pH [98, 99, 100]. Overall, the concentrations are low in the summer when metabolic activity is the highest and high in the winter when the metabolic activity is much lower [101, 102, 103].

Interestingly, all contaminants are taken up through different mechanisms, which also complicates the processing part that would be needed to remove the contaminants. Furthermore, within each contaminant class, the organic and inorganic forms also require different procedures to remove the respective forms. Effective removal of the contaminants is vital to ensure the safety of the seaweed consumption. Therefore, reduction methods should first be studied for each contaminant and sub-contaminant class, with a primary focus on the arsenic containing lipids, whereafter the procedures should be combined and optimized to create the shortest and most effective method to remove the contaminants.

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Chapter 3: Human bioavailability and metabolism of contaminants

. As stated in chapter 2, the S. latissima contains iodine and arsenic in various forms. After

ingestion, the seaweed undergoes several processes inside the human body that could alter its bioavailability. It is therefore important to assess which fraction of the contaminant species actually reaches the systemic circulation, especially since the bioavailability of the numerous species and metabolites could differ from one another.

Multiple in vitro studies are generally conducted to estimate the human bioavailability. The bioavailability is defined as the fraction of a compound that is able to enter the circulatory system after introduction to the body. To assess the bioavailability in vitro, the cell line of interest is incubated with the compound of interest (1-500 M). After a certain incubation period (24-72h), the accumulated internal concentration of the compound of interest is compared to the external concentration. The accumulation number then provides an indication on the bioavailability of that specific compound to the cell line of interest.

The cytotoxic potential of a compound is another important aspect that can be studied in vitro. The cytotoxicity provides a strong indication on the ability of the compounds to induce cell death and is generally estimated through the inhibitory concentration (IC) and effective concentration (EC), e.g. IC50 and EC50. The IC can be defined as the potency of a compound to

inhibit a specific biochemical or biological function and EC can be defined as the required concentration to obtain a desired effect. Both are generally assessed through the endpoints cell number, lysosomal integrity, dehydrogenase activity and colony forming ability. Similarly to the bioavailability, the cell lines are incubated with the compound (24 – 48h), where after the endpoints are studied through different types of assays. Additionally, the sensitivity of each endpoint to the compound of interest could provide additional information on its cytotoxic mode of action.

In vivo studies are another aspect that can provide important information with regards to the bioavailability and cytotoxicity. In vivo studies are generally conducted in living organisms that are comparable to humans, e.g. fruit flies or rats. In vivo studies also include human feeding studies, these studies are of great importance as the seaweed matrix could alter the bioavailability of the contaminants, moreover, it could also provide valuable information with regards to the metabolism of the contaminants. Therefore, this chapter will focus on the bioavailability and cytotoxicity of the contaminants in vitro, with a primary focus on the arsenicals and its metabolites, due to the severe toxic nature of these species. For this purpose the most relevant cell lines will be discussed and compared to one another. Finally, the chapter will also discuss relevant in vivo studies and will compare these studies to the discussed in vitro studies.

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Arsenic

Assessing the potential risks that arsenic species pose to the human body is very complex, especially since the biotransformation of the various arsenic species is not fully understood. As mentioned before, inorganic arsenic species are categorized as group 1, carcinogenic to humans by the International Agency for Research on Cancer (IARC) [104]. In addition to its carcinogenic properties, it is also associated with adverse effects such as skin lesions, neurotoxicity and cardiovascular diseases [105]. Upon exposure to inorganic arsenic through ingestion, the arsenic species are methylated and excreted from the human body into the urine. The excreted metabolites mainly consist of DMA(V) and MA(V), which have been identified as group 2B, possibly carcinogenic to humans by the IARC [104]. Studies have indicated that these methylated arsenic species are less toxic than the inorganic species. However, the potentially formed intermediates, DMA(lll) and MA(lll), are extremely toxic and pose great threat to the human body [106].

As stated in chapter 2, the primary arsenic content of the S. latissima consists of organic arsenic. Therefore, it is also important to assess the potential risks that these arsenicals and their metabolites pose to the human body. In Table 5 an overview is given of the identified arsenicals in the S. latissima and their identified or proposed metabolites after human ingestion. The table also highlights the current associated risks in terms of cytotoxicity. Similarly to inorganic arsenic, arsenic containing sugars and lipids are also primarily metabolized to DMA(V) after ingestion. In addition to DMA(V), thio-dimethylarsenopropanoic acid (thio-DMAP), thio-dimethylarsenobutanoic acid (thio-DMAB) and their oxo-analogs (oxo-DMAP, oxo-DMAB) were also identified as metabolites of the arsenic containing lipids, while dimethylarsinic acid (DMA(V)), dimethylarsenoacetic acid (DMAA(V)), thio-dimethylarsenoethanol (thio-DMAE(V)) and their oxo-analogs (oxo-DMAA(V), oxo-DMAE(V)) were identified as metabolites of arsenic containing sugars [107, 108, 109, 110, 111].

Table 5: Human bioavailability.

iAs(lll)1 _iAs(V)1 _AsB3 _AsSug

OH AsSug SO4 AsSug SO3 AsSug PO4

AsHC AsFA AsPL

MA(lll) P P P P P P P P P MA(V)2B _I _I _P _P _P _P _I _I _I DMA(lll) P P P P P P P P P DMA(V)2B _I _I _I _I _I _I _I _I _I Thio-DMA(V) P P P P P P P P P Oxo/thio-DMAE(V) I I I I Oxo/thio-DMAA(V) I I I I Oxo/thio-DMAP I I I Oxo/thio-DMAB(V) I I I TMAO I I I I AsB I Thio-AsSug I I I I Lit. [112, 113] [112, 113] [114] [110, 111] [110, 111] [110, 111] [110, 111] [107, 108] [107, 108] [107, 108] IARC classification: Legend:

1 _{Carcinogenic to humans} _{Has not been proposed or identified as metabolite} 2B _{Possibly carcinogenic to humans}

Parent compound / metabolite that is bioavailable but exerted no cytotoxic effects in vitro

3 _{Not classifiable as to its carcinogenicity to humans}

Parent compound / metabolite that is bioavailable and exerted cytotoxic effects in vitro

I Identified metabolite in human urine Parent compound / metabolite that is bioavailable and exerted cytotoxic effects in vitro and in vivo

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As of yet, the metabolites thio-DMAA(V) and thio-DMAE(V) have only been associated with the metabolism of arsenic containing sugars. It was therefore suggested that these thiolated arsenic species could provide unique markers of the arsenic containing sugar metabolism and thereby distinguish arsenic originating from seaweed with respect to other arsenic sources, such as drinking water [115]. The formation of DMA(V) alludes to the possibility that the biotransformation of arsenic containing lipids and sugars is comparable to that of inorganic arsenic. The intermediates that are formed during the conversion of inorganic arsenic to DMA(V) are currently considered to play a role in its cytotoxic mode of action [116]. It is likely that during the metabolization of the organic arsenicals similar intermediates are formed, this raises the issue on whether these organic arsenicals are also capable of producing toxic effects to the human body. These arsenic species should therefore be treated with great caution before cytotoxic activity in the human body is ruled out.

The following section will evaluate the bioavailability and toxic nature of inorganic arsenic (iAs(lll)), arsenic containing sugars (AsSug-SO4, AsSug-OH, thio-AsSug-OH), arsenic containing lipids (AsHC 332, AsHC 360, AsHC 444, AsFA 362, AsFA 388) and important metabolites. The section will focus on the in vitro analysis of different cell lines and conducted in vivo studies. The section will discuss the bioavailability and cytotoxicity in the order that the arsenic compounds of the seaweed would normally move through the human body, see Figure 14.

Figure 14. Order in which the arsenic compounds of the seaweed would move through the human body [117_].

Seaweed consumption

Gastrointestinal tract

Feces Intestinal _barrier

Liver Bladder Urine Circulatory system Blood brain barrier Brain

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Arsenicals in the gastrointestinal tract

After ingestion of arsenic containing seaweeds, the arsenic containing compounds enter the gastrointestinal tract, see Figure 14. Inside the gastrointestinal tract, the intestinal barrier regulates the absorption of nutrients and protects the circulatory system from non-desirable compounds [118]. Epithelial cells are an important component of the intestinal barrier and are therefore suitable to indicate whether the arsenic species are able to pass the barrier and enter the body [119]. The epithelial cells contain an apical compartment and a basolateral compartment. The apical compartment, also referred to as apical side or apical membrane, is the cell membrane that is oriented towards the lumen, while the basolateral compartment or basolateral side/membrane is oriented away from the lumen and faces the blood side [120].

In vitro studies

In vitro studies are a suitable method to provide an indication on which arsenic fraction is able to pass the intestinal barrier and which fraction is excreted via the feces. For this purpose, various in vitro studies have recently been conducted in an intestinal barrier model using human epithelial cells (Caco-2). These studies evaluated the bioavailability, presystemic metabolization and cytotoxicity of iAs(lll), AsHCs, AsFAs, AsSug-OH and AsSug-SO4 and its

metabolites. For an overview of the gathered raw data see Appendix 7 and Appendix 8.

Bioavailability in vitro

Results showed that all studied AsHCs and AsFAs were bioavailable to the Caco-2 cells, see Figure 15A. The transfer of AsHC 332 was most efficient. Furthermore, the transfer of all AsHCs towards the apical compartment was less efficient than the transfer towards the basolateral compartment. These results allude to a potential transfer of up to 50% across the intestinal barrier [121]. It is likely that the remainder is excreted via the feces. However, these numbers are based on the assumption that the arsenic components pass the intestinal barrier intact, while it is also possible that the compounds undergo presystemic metabolization inside the GIT, which will be discussed later.

In contrast to the arsenic containing lipids, the transfer of the arsenic containing sugars across the Caco-2 cells was almost non-existent, see Figure 15A [131, 132]. Nevertheless, it is still possible that these arsenic compounds are presystemically metabolized in the GIT and therefore could enter the system in its metabolized form, e.g. DMA(V), DMA(V), thio-DMAE(V). The transfer of three out of the four reviewed metabolites was comparable to the transfer of the arsenic containing sugars, see Figure 15A. Therefore, it is likely that these arsenic compounds pose little threat to the human health, as the largest fraction would be excreted from the body via the feces. However, the transfer of the fourth studied metabolite, thio-DMAE(V), was comparable to the transfer of iAs(lll), see Figure 15A [122].

Unfortunately, the presystemic metabolization of the arsenic containing sugars, as well as, the transfer of the primary metabolites DMA(V) and thio-DMA(V) has not yet been studied. Furthermore, the cytotoxicity of the arsenic containing sugars and metabolites to the Caco-2 cells has also not yet been studied, which makes it difficult to assess the risks with respect to these arsenic species.

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Cytotoxicity in vitro

Results indicated that all three AsHCs exerted cytotoxic effects to the Caco-2 cells, as the endpoints lysosomal integrity and dehydrogenase activity were both affected. At the highest incubated concentration (100 M) AsHC 360 showed the strongest effects, nevertheless, AsHC 332 was proposed as the most cytotoxic AsHC with respect to the EC70 values, see Figure 15B.

The studied AsFAs did not exert cytotoxic effects up until the highest incubated concentration (100 M) [121]. Furthermore, results indicated that thio-DMA(V) exerted cytotoxic effects in the same micromolar concentration range as iAs(lll), see Figure 15C. Moreover, both endpoints were most sensitive to thio-DMA(V) [132].

Figure 15 Bioavailability and cytotoxicity arsenicals to in vitro model Caco-2 cells. A: Transfer arsenicals across Caco-2 cells. B: Cytotoxicity with endpoint EC70. C: Cytotoxicity with endpoint IC70. Only arsenicals that reached the endpoint are displayed in the figure. Figure based on data of Meyer et al. 2015 [121], Leffers et al. 2013a [132], Leffers et al. 2013b [122], Ebert et al. 2016 [131].

0 50 100

AsHC 360 AsHC 444 AsHC 332

EC

7

0

Cytotoxicity Caco-2 cells

Lysosomal integrity Dehydrogenase activity

0 10 20 30 40 Thio-DMA(V) iAs(lll) IC 7 0

Cytotoxicity Caco-2 cells

Lysosomal integrity Dehydrogenase activity 1 10 100 Tra ns fer (% )

Bioavailability Caco-2 cells

Transfer towards apical compartment Transfer towards basolateral compartment

A

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Presystemic metabolization in vitro

The toxicity is different for each arsenic species and it is therefore of great importance to understand in which form the arsenic species enters the circulatory system. Presystemic metabolization essentially means that the concentration of the parent compound is greatly reduced before it reaches the systemic circulation, thus also the bioavailability of the parent compound is reduced.

The presystemic metabolization of the AsHCs in Caco-2 cells varied between 3-14%. The primary metabolites included arsine (AsH3) and thio-analogs. Contrary to the AsHCs, the AsFAs were almost completely metabolized as only 5-13% was identified as the parent compound. The metabolites primarily consisted of polar arsenic species, of which DMA(V) was the only metabolite that could be identified [121]. Furthermore, results showed that iAs(lll) was not metabolized. Contrary to iAs(lll), 31% of the thio-DMA(V) was metabolized into DMA(V), the remaining fraction was identified as the parent compound [122].

The conducted in vitro studies suggest that the AsHCs and AsFAs are bioavailable to the Caco-2 cells. Therefore, it is likely that these arsenic species are able to pass the intestinal barrier in the human body. Results also suggested that the AsHCs were able to pass the membrane intact, while the AsFAs were largely presystemically metabolized. Unfortunately, the transfer and cytotoxicity of its primary metabolite, DMA(V), was not studied. Furthermore, these in vitro studies only focused on the bioavailability of the pure arsenic compounds. In reality, the arsenic compounds enter the human body within a seaweed matrix. This could significantly affect the bioavailability of the arsenic compounds, which will be discussed in the following section.

In vivo studies

In 2017, an in vivo feeding study was performed on human volunteers. During this study the bioavailability of the brown seaweed Laminaria japonica (Kombu), the red seaweed Porphyra

umbilicalis (Nori) and the green seaweed Undaria pinnatifida (Wakame) was studied. Results

suggested that the bioavailability of Nori (7.1-48.3%) was the highest, this was followed by Wakame (9.7-29.5%) and Kombu (1.4-19.6%), see Figure 16A. Furthermore, speciation analyses was conducted on the urine of the volunteers to identify the metabolites.

The study revealed that the magnitude in which the metabolites were present in the urine was the same for all studied seaweeds with DMA(V) as the primary metabolite, followed by DMAA(V), DMAE(V), AsSugs and DMA(V), see Figure 16B. As stated before, thio-DMAA(V) and thio-DMAE(V) have only been associated with the metabolism of arsenic containing sugars and therefore also highlight the bioavailability and metabolism of the arsenic containing sugars, this is an important revelation as the in vitro studies mainly showed low bioavailability’s with respect to the arsenic containing sugars [115]. As stated above, the in vitro studies only studied the bioavailability of the intact AsSug species. Therefore, it is likely that the observed differences with respect to the bioavailability in vitro and in vivo are caused by the presystemic metabolization of the intact AsSug species upon entering the GIT. This means that the species enter the circulatory system in a different arsenic form, which accordingly has a different bioavailability.

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An important factor that is highlighted in this feeding study is the individual variability with regards to the metabolization of the arsenic species. As of today, these variabilities remain unexplainable, as it could not be attributed to gender, BMI or age. A different feeding study even showed that the arsenic excretion after pure arsenic containing sugar ingestion varied from 4 to 95%, as two out of the six individuals excreted less than 15% of the ingested arsenic in the urine [123].

The current proposed hypothesis for these observed differences is the potential individual differences in the arsenic absorption or breakdown in the gut. Even though the absorption and metabolic pathways of organic arsenic is not fully known, the current theory implies that part of the arsenic is absorbed in the gut, whereafter the majority is excreted via the kidneys in the urine. The part of the arsenic that is not absorbed is then excreted in the feces [124, 125]. Factors that could contribute to the individual variability are differences in the gut microbiome composition, genetic differences that affect enzyme production or differences in lifestyle [115]. However, more studies are needed to confirm this theory.

Even though the study did not imply the seaweed of interest, S. latissima, it does provide an outlook on the human bioavailability and metabolism of arsenic originating from seaweed. Nevertheless, future studies are needed to assess the bioavailability from the S. latissima, as the seaweed matrix could alter its bioavailability.

Figure 16 Human feeding study. A: Bioavailable total arsenic fraction, placed on a logarithmic scale. B: Identified metabolites in human urine, placed on a logarithmic scale. Figure based on data of Taylor et al. 2017 [115].

Arsenicals in the liver

The arsenic compounds that pass the intestinal barrier of the GIT enter the liver, where they could be metabolized into different arsenic species, see Figure 14 [115]. Hereafter, the arsenic compounds are either excreted via urine or released into the systemic circulation of the body. In vitro studies could provide a great indication on whether arsenic species are metabolized in the liver and if they pose any threat to human liver cells. Over the years, various in vitro studies evaluated the bioavailability and cytotoxicity of iAs(lll), AsHCs, AsFAs, thio-AsSug-OH and important metabolites to human liver cells (HepG2 cells). For an overview of the gathered raw data see Appendix 7 and Appendix 8.

1 10 100

Nori Kombu Wakame

TA s (% ) Bioavailable fraction 0.01 0.1 1 10 100 TA s (% )