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European Epi-Marker Vol. 11, No. 3 :1-12. July 2007 The Newsletter of the International Center for Studies and Research in Biomedicine, Luxembourg

Evolution of dietary antioxidant defences Sebastiano Venturi and *Mattia Venturi Servizio di Igiene, ASL n. 1, Regione Marche, Pennabilli (PU) Italy, and *Department of Oral Science, University of Bologna, Italy

Corresponding address: Dr. Sebastiano Venturi - via Tre Genghe n. 2; 61016-Pennabilli (PU), Italy Tel : (+39) 0541 928205 . E-mail: [email protected]

KEY WORDS :

Antioxidants, ascorbic acid, carotenoids, flavonoids, evolution, iodine, selenium;

SUMMARY The evolution of oxygen-producing cells was probably one of the most significant events in the history of life. Protective endogenous antioxidant enzymes and exogenous dietary antioxidants (as Selenium, Iodine, etc.) helped to prevent oxidative stress. Iodide, which acts as a primitive electron-donor through peroxidase enzymes, seems to have an ancestral antioxidant function in all iodide-concentrating cells from primitive marine algae to more recent terrestrial vertebrates. When about 500 million years ago plants and animals began to transfer from the sea to rivers and land, environmental iodine-deficiency was a challenge to the evolution of terrestrial life. New endogenous antioxidants appeared in plants as

ascorbic acid, polyfenols, carotenoids, flavonoids. A few of these appeared recently, about 200-50 million years ago in fruits and flowers of angiosperm plants. In the wide range of antioxidants, we hypothesise an “evolutionary hierarchy”, where the most ancient antioxidants might be more essential than the modern ones in the developing stages of animal and human organisms.

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INTRODUCTION Oxygen is a potent oxidant whose accumulation in terrestrial atmosphere began as result of the development of photosynthesis, over three billion years ago, in blue-green algae (Cyanobacteria), which were the most primitive oxygenic photosynthetic organisms. In this review, we discuss different evolutionary strategies of antioxidant defence mechanisms in plants and in animals. In general terms, an antioxidant is anything which can prevent or inhibit oxidation (Benzie, 2000). This can be achieved by preventing or by inactivating the generation of reactive oxygen species (ROS). We review the evolution of some antioxidants: a) in particular, Iodide/Iodine and Iodide/Thyroxine; and more briefly: b) Selenium; c) Ascorbic acid; d) Carotenoids and Flavonoids. The second aim of this paper is to provide a medical perspective. In fact, the importance of antioxidants as protective substances against many chronic and degenerative diseases, such as cancer, cardiovascular and oral diseases, is very well-known. But the utility of well-known antioxidants in these diseases has not been recently supported by statistical data and their utility in cancer and atherosclerotic disease prevention has not been recently confirmed by epidemiological data (Bjelakovic et al., 2004, 2007, 2008; Hung et al., 2004; Lin et al., 2005; Sato et al ., 2005; Tsubono et al., 2005; Venturi et al., 2000; Aceves et al., 2005; Morris and Carson, 2003). In the wide range of antioxidants, we hypothesise an “evolutionary hierarchy” of these substances, where the most ancient might be more essential than the modern ones in the developing stages of animal and human organisms. Deficiency of iodine, as a primitive antioxidant, causes more damage in developing embryos ( Dunn and Delange, 2001 ) than some other “modern” antioxidants. In fact, in pregnant women I-deficiency causes abortions and stillborns. This damage seems not completely caused by thyroid hormones (TH) deficiency, but above all by iodine deficiency “per se” (Wolff, ’64; Goethe et al. ’99).

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Increase of Oxygen in Earth’s Atmosphere and its Biological Consequences The evolution of oxygen-producing cyanobacteria was probably the most significant event in the history of life after the evolution of life itself. Oxygen is a potent oxidant whose accumulation into the atmosphere forever changed the surface chemistry of Earth (Canfield, 2005). Oxygen may have stimulated diversification as well as extinction of life (Lane, 2002). It is currently believed that the oxygen concentration in Earth's atmosphere may have remained at 1 % of its present level until approximately 2 billion years ago, after which the concentration gradually increased to its present value (about 21 %) with the increasing success of photosynthetic life forms (Lane, 2002, 2005). Although the time-frame of the increase in oxygen concentration of the atmosphere is uncertain, the consensus among researchers is that the initiation of an oxygen atmosphere increased the number and kinds of organisms capable of using aerobic metabolic pathways. Wiedenheft et al. (2005) and Benzie et al. (2003) suggested that the evolution of oxygenic photosynthesis marks the dawn of oxidative stress and represents one of the greatest selective pressures imposed on primordial life. The association of molecular oxygen with abundant ferrous iron pools created two major biological consequences. First, life dependent on the redox properties of Fe(II) would have to contend with its oxidation and precipitation as Fe(III). Second, life would have to contend with the toxicity of ROS generated by the partial reduction of dioxygen by ferrous iron. Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms. By the start of the Cambrian period 570 million years ago, or somewhat earlier, oxygen levels had apparently increased enough to permit rapid evolution of large oxygen-utilizing multicellular organisms. Then oxidation processes in the cellular metabolism became essential to generate the energy necessary for the life of higher aerobic species. However, during these processes also highly ROS and reactive nitrogen species (RNS) are produced. These reactive molecules potentially react with lipids, proteins, carbohydrates and DNA and thus interfere with the functions of cellular membranes, cell metabolism, cellular signalling, cell growth and differentiation. During evolution, endogenous protection systems have developed to counteract the deleterious effects of cellular oxidation. Protective antioxidant enzyme systems consist primarily of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase and peroxiredoxins. In addition to these endogenous systems, exogenous dietary antioxidants may help to prevent oxidative stress. In particular, mineral antioxidants of marine origin, present in primitive sea, as some reduced compounds of Rubidium, Vanadium, Zinc, Iron, Cuprum, Molybdenum, Selenium, Iodine (I), etc. which play an important role in electron transfer and in redox chemical reactions. Most of these substances act in mammals as essential trace-elements in redox and antioxidant metallo-enzymes. Iron, for example, present in 4

5 many iron-porphyrins, can be a pro-oxidant if it donates electrons to molecular oxygen (thus forming ROS). Alternatively, iron can be an antioxidant if it donates electrons to ROS and forms water. In this way, iron-porphyrins play a vital role in many biological functions including oxygen transport, electron transfer and catalyzing the incorporation of oxygen into other molecules (Chappuis, ‘91). Some researchers hypothesized that the relative composition of many mineral trace-elements of the animal body is similar to the composition of the primitive sea, where the first forms of life began (Favier, ’91). Stone (1988) studied the role of the primitive sea in the natural selection of iodides as a regulating factor in inflammation. This author reported that iodides have many non-endocrine biologic effects, including a role they play in the physiology of the inflammatory response. Iodides increase the movement of granulocytes into areas of inflammation and improve the phagocytosis of bacteria by granulocytes and the ability of granulocytes to kill bacteria. Oxidative stress has been implicated as a causative process in the development of a vast number of degenerative diseases (Suzuki et al., ’97; Flohe et al., ’97; Yu, ’94; Sies, ’97). The reactions of ROS with DNA and the resulting oxidative modification of purin or pyrimidin bases may lead to mutations. These mutations, if not corrected through specific DNA repair mechanisms, may finally lead to carcinogenesis. Successively antioxidants of terrestrial origin appeared in plants as many of polyfenols, carotenoids and flavonoids etc. A few of these appeared more recently, about 200-50 million years ago (Mya) in fruits and flowers (Venturi, 2004).

Early developments in antioxidant defence Several types of antioxidant mechanisms exist, and these are outlined in Table 1 (from Benzie, 2000). The first antioxidant mechanisms were probably simple physical barriers. Intracellular sequestration of photosensitising pigments, such as chlorophyll, and compartmentalisation of vulnerable cellular components, would serve to protect against the production and action of ROS.

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Table. 1. (Reported from Benzie, 2000)

Oxygen is vital for most organisms but, paradoxically, damages key biological sites. Oxygenic threat is met by antioxidants that evolved in parallel with our oxygenic atmosphere. Plants employ antioxidants to defend their structures against ROS produced during photosynthesis. The human body is exposed to the very same oxidants, and it has also evolved an effective antioxidant system. Plant-based, antioxidant-rich foods traditionally formed the major part of the human diet, and plantbased dietary antioxidants are hypothesized to have an important role in maintaining human health. Benzie (2003) reported that also estimated daily intake of many selected antioxidants (such as antioxidant vitamins, carotenoids etc.) decreased quantitatively from Palaeolithic to modern human diets. Effective chemical antioxidants may be very stable in their oxidized form (e.g., glutathione), or part of a larger system for "chain breaking" the cycle of oxidation and reduction, or typically both. Iodide/thyroxine is one such "chain-breaking" system, where the oxidized product (iodine) is safely sequestered and thus cannot oxidize other molecules.

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Iodide/iodine and Iodide/thyroxine: Evolutionary history of a primitive antioxidant 2 I-
I2 + 2 e- (electrons) = - 0.54 Volt ; 2 I- + Peroxidase + H2O2 + 2 Tyrosine
2 Iodo-Tyrosine + H2O + 2 e- (antioxidants); 2 e- + H2O2 + 2 H+ (of intracellular water-solution)


2 H2O

Table. 2. Proposed antioxidant biochemical mechanism of iodides (From Venturi, 1985).

2 I- + Peroxidase + H2O2 + Tyrosine, Histidine, Lipids, Carbons

Iodo-Compounds + H2O + 2 e- (antioxidants) Iodo-Compounds: Iodo-Tyrosine, Iodo-Histidine, Iodo-Lipids, Iodo-Carbons Table. 3. Proposed antioxidant biochemical mechanism of iodides, probably one of the most ancient mechanisms of defence from poisonous reactive oxygen species (Modified from Venturi, 2003).

Over three billion years ago, blue-green algae (Cyanobacteria), were the most primitive oxygenic photosynthetic organisms, ancestors of multicellular eukaryotic algae. Algae that contain the highest amount of iodine (1-3 % of dry weight) and peroxidase enzymes, were the first living cells to produce poisonous oxygen in the atmosphere (Obinger et al., ‘97a, b; Venturi et al. 2000a, b). Therefore we suggest that algal cells required a protective antioxidant action of their molecular components, in which iodides, by peroxidase enzymes, seem to have had this specific role (Venturi ’85; Venturi et al., ’87; Venturi et al., ‘93; Venturi and Venturi ‘99). In fact iodides are greatly present and available in the sea, where algal phytoplankton, the basis of marine food-chain, acts as a biological accumulator of iodides, selenium (and n-3 fatty acids) (Cocchi and Venturi, 2000 ). The sea is rich in iodine, about 60 micrograms (µg) per litre in coastal seawaters, since this is where most of the iodine removed and washed away from the soil accumulated by rains and by the glacial ages. In the open ocean the total iodine concentration is around 0.5 mM or 0.06 ppm (Elderfield and Truesdale, ’80). The major iodine species in coastal sea waters are iodate (IO3-) and iodide (I-), along with smaller concentrations of molecular iodine, hypoiodous acid and iodinated organic compounds (Truesdale et al., ’95). Brown algae (seaweeds) accumulate iodine to more than 30,000 7

8 times the concentration of this element in seawater, up to levels as high as 1-3 % of dry weight (Colin et al. 2003; Teas et al., 2004). Not much is known, however, on the iodine-concentrating mechanisms and on the biological functions of iodine in algae. Primitive marine prokaryotes seem to have an efficient active “iodide pump”, ancestor of the pump of multicellular eukaryotic algae and of mammalian iodide transporters. The mechanism of iodide-pumps in the cells is very ancient and lacking of specificity, in fact, it is not able to distinguish iodide from other anions of similar atomic or molecular size, which may act as “pseudo-iodides”: thiocyanate, cyanate, nitrate, pertechnate, perchlorate, etc. (Wolff, ’64). Algae, cyanobacteria and prochlorophytes are primary producers in sun-lit aquatic (and some terrestrial) food chains fixing CO2 into organic molecules through photosynthesis (photoautotrophy) that can be consumed by chemoheterotrophic organisms. Molecular oxygen (O2) is a by-product of oxygenic photosynthesis and it is hypothesized that 80% of the Earth's oxygen is produced by planktonic algae, prochlorphytes and cyanobacteria, the freefloating unicellular microbes that inhabit the top few meters of water. Up till now only one aspect of halogen metabolism, the production of volatile halocarbons, seems to have attracted more attention from researchers, because these compounds, and in particular the iodinated forms, have a significant impact on the chemistry of the atmosphere, and its ozone shield depletion (Carpenter et al., ’99; Carpenter et al., 2000). Marine macroalgae are known to emit volatile halo-carbons naturally, including a variety of short-lived organo-iodines, which are believed to be a main vector of the iodine biogeochemical cycle as well as having a significant impact on the oxidant levels of the troposphere (Carpenter et al., ’99; Carpenter et al., 2000). Halogen metabolism in marine algae involves enzymes known as haloperoxidases, which catalyse the oxidation of halides into hypohalous acids (Vilter et al., ’83; Vilter, ’94; Vilter, ’95; Gribble, ’96; Pedersèn et al., ’96; Dembitsky et al., 2003; Gall et al., 2004). Since iodoperoxidase of Laminaria seaweeds is more efficient than the bromoperoxidase in the oxidation of iodide (Colin et al. 2003), this former activity may be more largely responsible for the uptake of iodide from seawater. There is an increased emission of iodinated halocarbons both from kelp beds at low tide during day-time (Carpenter et al., ’99; Carpenter et al., 2000), and from kelp plants incubated under high solar irradiance, caused by photo-oxidative stress, compared to plants kept in the shade (Gall et al., 2004). The green algae are hypothesized to have been ancestors of terrestrial plants. In 1985, Venturi suggested that iodide might have an ancestral antioxidant function in all iodide-concentrating cells from primitive marine algae to more recent terrestrial vertebrates (Venturi, ’85; Venturi, ’93; Venturi et. al., 2000a, b; Venturi, 2001). In these cells iodide acts as an electron donor in the presence of hydrogen peroxide and peroxidase, and the remaining iodine atom readily iodinates tyrosine, histidine or certain specific lipids. The production of these iodo-compounds (Iodo-Tyrosine, Iodo-Histidine, Iodo8

9 Lipids, Iodo-Carbons etc.) was probably one of the most ancient mechanisms of defense from primitive poisonous ROS (Venturi, ’85; Venturi, ’93; Venturi et. al., 2000a, b; Venturi, 2001). In vertebrates, isolated cells of extrathyroidal iodide-concentrating tissues can produce protein-bound mono-iodo-tyrosine (MIT), di-iodo-tyrosine (DIT) and also some iodolipids (Banerjee et al., ’85; Aceves et al., 2005). Iodolipids have been shown to be regulators of mammalian cellular metabolism. This second pathway for iodine organification has been described, which involves iodine incorporation into specific lipid molecules. Iodine reacting with double bonds of some polyunsaturated fatty acids of cellular membranes make them less reactive with free oxygen radicals (Cocchi and Venturi, 2000). Two iodinated lipids, synthesized by the thyroid gland, may be iodine autoregulation mediators: 6-iodo-5-hydroxy-8,11,14-eicosatrienoic acid (delta-iodo-lactone) and 2-iodohexadecanal. Delta-iodolactone has been found to be a potent inhibitor of proliferation of thyroid and of some non-thyroidal cells (Banerjee et al., ’85; Pisavev et al., ’88; Dugrilllon, ’96; Venturi et al., 2000a, b; Cocchi and Venturi, 2000; Cann et al., 2000; Aceves et al., 2005). According to Aceves et al. (2005) the percentage of iodine in cellular homogenate of breast tissue is about 40 % in lipid fraction and 50 % in protein fraction. Aceves also reported that in mammary gland homogenates from virgin rats, the addition of iodine in their diet significantly decreases lipid peroxidation. Petersén et al. (1996), Küpper et al. (1998; 2002) and Gall et al. (2004) suggested that the production of volatile iodo-compounds by marine algae is a result of the development of photosynthesis, oxygen production and respiration some 3 billion years ago, and it is due to adaptation to light in order to reduce the amount of poisonous ROS, such as hydrogen peroxide, superoxide radicals and hydroxyl radicals. The algae defence system is an enzymatic removal system, built on peroxidases and catalases (Küpper et al. ‘98, 2002 ; Petersén et al., 1996). In the seaweeds, iodine is believed to be localised in non-specialised vacuoles in the blade cortical cells and in phenol-containing physodes in stipe cells (Amat, ’85; Amat and Srivastava, ’85). It is mainly stored in the form of inorganic iodine, which amounts to 80–90% of the total iodine content, the remainder consists of the iodinated amino-acids, mono- and di-iodo-tyrosine (Roche and Yagi, ’52). The family of peroxidase enzymes includes mammal, microorganism, plant, algal, and fungal peroxidases. Some of these peroxidases, known as haloperoxidases, use halide ions (iodide, bromide, and chloride) as natural electron donors, and have an antioxidant function in Cyanobacteria (Obinger et al., ’97; Obinger et al., ’99; Venturi and Venturi, ’99 ). Taurog (1999) reported that the relation between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism. Heyland and Moroz (2005) suggest that exogenous sources of thyroid hormones (THs) (from food) may have been ancestral, while the ability to synthesize TH endogenously may have evolved independently in a variety of metazoans, 9

10 resulting in a diversity of signaling pathways and, possibly, morphological structures involved in TH-signaling. In fact, increasing evidence suggests that THs also function in a variety of invertebrate species. The evidence of TH effects in invertebrates has been reviewed in Eales (’97) and Heyland et al. (2005).

Evolution of Selenium, Ascorbic Acid, Carotenoids and Flavonoids Selenium Selenium is an antioxidant mineral, greatly present in primitive seawaters and is an essential component of the enzyme glutathione peroxidase in mammals. Selenium is present in cellular peroxidases and deiodinases ( type 1 and 3), which are able to extract electrons from iodides, and the latter iodides from iodothyronines. One family of selenium-containing molecules as glutathione peroxidases (GPx) repairs damaged cell membranes, while another (glutathione S-transferases) repairs damaged DNA and prevents mutations ( Stadtman, ’96). Selenium acts synergistically with iodine. The mono-deiodinase ( type I and type III ) enzymes are selenium-dependent and are involved in thyroid hormone regulation. In this way selenium status may affect both thyroid hormone homeostasis and iodine availability. Several selenoproteins participate in the protection of thyrocytes from damage by H2O2 produced for thyroid hormone biosynthesis. The deiodinase isoenzymes constitute the second family of eukaryotic selenoproteins with identified enzyme function. Deiodinases catalyze the reductive cleavage of aromatic C-I bonds in ortho-position to either a phenolic or a diphenylether oxygen atom in iodothyronine. Trace elements involved in glutathione peroxidase and superoxide dismutases enzymes activities – i.e. selenium, magnesium, copper and zinc, may have been lacking in some terrestrial iodine-deficient areas (Gladyshev and Hatfield, ’96). From about three billion years ago, prokaryotic selenoprotein families drive selenocysteine evolution. This micronutrient is incorporated into several prokaryotic selenoprotein families in bacteria, archaea and eukaryotes as selenocysteine (Gladyshev and Hatfield, ’96). The selenoprotein peroxiredoxins protect bacterial and eukaryotic cells against oxidative injury. Also selenoprotein families of GPx and deiodinase of eukaryotic cells seem to have a bacterial phylogenetic origin. The selenocysteine-containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. New selenoproteins are recently (about 200 Mya) developed in 10

11 mammalians (Castellano et al., 2004; Kryukov and Gladyshev, 2004; Wilting et al. ’97; Zhang et al., 2005). Marine fish and vertebrate thyroid glands have the highest concentration of selenium and iodine.

Ascorbic acid (Vitamin C) Ascorbic acid (“Vitamin C” for humans) is a common antioxidant in mammals . Ascorbic acid is a water soluble antioxidant and it exists primarily as ascorbate at physiological pH. Ascorbate is a powerful reducing agent capable of rapidly scavenging a number of ROS. Freshwater teleost fishes also require dietary (vitamin C) in their diet or they will get scurvy (Hardie et al.,’91). The most widely recognized symptom of Vitamin C deficiency in fish is scoliosis, lordosis and dark skin coloration. Terrestrial freshwaters salmonids also show impaired collagen formation, internal/fin haemorrhage, spinal curvature and increased mortality. If fishes are housed in seawater with algae and phytoplankton, then vitamin supplementation seems to be less important, presumably because of the availability of other antioxidants in natural marine environment (Hardie et al.,’91). For this reason, we suggested that the antioxidant action of ascorbic acid developed firstly in the plants when, about 400 Mya, plants (and animals), began to adapting themselves to mineral (and iodine) deficient fresh waters of estuary of rivers and land. In fact, some biologists suggested that many vertebrates had developed their metabolic adaptive strategies in estuary environment (Purves et al., ‘98). Like plants, most mammals (with the exception of humans and guinea pigs) make their ascorbic acid from glucose and can make glucose from ascorbic acid. Remote ancestors of humans suffered a genetic mutation about 40-45 million years ago and haven't been able to make “vitamin C” since. Therefore living humans need nowadays to get all our “vitamin C” from food. Some scientists think that the loss of our ability to make “Vitamin C” may have caused Homo Sapiens' rapid evolution into modern man (Challen et al., ’98; Benhegyi et al., ’97; Stone, ’79).

Fig. 1 . Cultured salmons in freshwater showing nutritionally induced spinal curvature (scoliosis and lordosis) by vitamin C deficiency. 11

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Carotenoids and Flavonoids More than 600 plant carotenoids have been identified in plants but less than 50 are abundant in the human diet. Carotenoids are nutritionally important for normal cell regeneration, plus numerous other health aspects linked to ROS (Packer, ’92; Packer, ’93; Cadenas and Packer, 2002). Lucy and Lichti (1969) reported an interesting electron transfer from vitamin A (and carotenoids) to iodine in cellular membranes, in which iodide ions are formed, where a double bond in retinol (or retinoic acid) is presumably the source of these electrons that are transferred from the vitamin to iodine. Two different mechanisms may be proposed in the present context to account for the effectiveness of very small quantities of vitamin A in vivo. (1) The electron-deficient products that are formed when the vitamin behaves as a donor have access to a regenerative source of electrons. (2) Vitamin A interacts with a donor of electrons immediately before, or at the same time as, it donates electrons itself. The electron-acceptor properties of vitamin A, and its interactions with electron-donor substances, may be relevant to the latter possibility. In both of these theoretical mechanisms electron transfer occurs without the vitamin being destroyed. In fact carotenoids and vitamin A (as iodine/iodide) are both excellent donors and acceptors of electrons. Vitamin A participates catalytically in certain oxidation-reduction reactions that are essential for the normal functioning of organized tissues in vivo, and the physical properties of vitamin A indicate that these reactions may occur on or in membranes. Flavonoids and carotenoids are the biggest group of antioxidant phytochemicals studied. Some of these pigmented antioxidants form the colours of plants and in particular of fruits and flowers. Flowering plants are Angiosperms, which produce seeds enclosed in fruits and flowers. Angiosperms, the dominant type of plant today, evolved rather recently about 200-50 million years ago from cone-bearing gymnosperms, during the late Jurassic period. So also most of the antioxidant pigments have evolved rather recently. Most carotenoids are "sacrificial" antioxidants, in other words, carotenoid molecules are not regenerated like other antioxidants, and are degraded in the process of neutralizing ROS.

Evolution of iodine from non-hormonal to hormonal functions Iodide uptake is present in primitive Cyanobacteria and in algae, plants, Porifera, Anthozoa, and arthropods without showing any hormonal or biological action. Since approximately 700-800 Mya thyroxine (T4) has been also present in fibrous exoskeletal scleroproteins of the lowest marine 12

13 invertebrates (sponges, corals etc.) ( Roche, ’52). Recent studies reported that thyroid hormones are also present in unicellular planktonic alga (Dunaliella tertiolecta) and in echinoid larvae (sea-urchin) (Chino et al., ’94; Heyland, 2004). These original sources of animal hormones might have been plants/algae in many cases, and could well have been independently derived from plants/algae in distinct lineages. The ancestral function of thyroid hormone could also have been as feeding deterrents in algae and/or plants and the signalling functions in animals (Heyland and Moroz, 2005; Eales, ’97) might, therefore, have been acquired secondarily, perhaps even through horizontal transfer from their hosts or other co-associated microbes with more ancient relationships with the host. In waters the iodine concentration decreases step by step from sea-water to estuary (about 5 µg / L) and source of rivers (less than 0.2 µg / L in some Triassic mountain regions of northern Italy), and in parallel, salt-water fishes (herring) contain about 500-800 µg of iodine per kg compared to fresh-water trouts about 20 µg per kg (Venturi and Venturi, ‘99; Venturi et al., 2000a, b; Venturi et al., 2003). So, in terrestrial iodine-deficient fresh waters some trout and other salmonids (anadromous migratory fishes) may suffer thyroid hypertrophy or related metabolic disorders (Venturi et al., 2000a, b), as do some sharks in captivity. Youson and Sower (2001) reported that iodide-concentrating ability of the endostyle of sea lamprey was a critical factor in the evolution of metamorphosis and that the endostyle was replaced by a follicular thyroid, since postmetamorphic animals needed to store iodine following their invasion of freshwater. According to Manzon and Youson (1997) in some anadromous migratory fishes (sea lamprey and salmonids), iodine and thyroid hormones (TH) play a role in initiation of metamorphosis, which is induced by the decline in serum of TH. After metamorphosis, when these adult marine fishes die in fresh water after reproducing, they release their iodides and selenium, and n-3 fatty acids (Venturi et al., 2000a, b), in the environment, where they have a favourable role in food for life and health of native animals, bringing back upstream from the sea to iodine-deficient areas a considerable quantity of these trace-elements (Venturi et al., 2000a, b). Broadhurst et al. (2002) and Cunnane (2005) suggested that early Homo sapiens, living around the Rift Valley lakes and up the Nile Corridor into the Middle East, received iodine and n-3 fatty acids from littoral food resources. The role of iodine in marine and fresh-water fishes has not been completely understood, but it has been reported that iodine-deficient fresh-water fishes suffer of higher incidence of infective, parasitic, neoplastic, and atherosclerotic diseases than marine fishes. Farrell et al. (1992) reported the absence of coronary arterial lesions in some elasmobranch fishes, living in iodine-rich sea-water. The discovery that coronary atherosclerotic lesions are present in migratory fresh-water salmon and yet appear to be absent in marine sharks raises interesting questions regarding the etiology of some type of arteriosclerosis. In October 7, 1999, the U.S.A. Committee of the House and Senate regarding 13

14 "Marine Research" reported that " The Committee notes the unusually low incidence of cancer in marine sharks, skates, and rays and encourages basic research through the study of the immune system of these marine animals and the examination of bioactive molecules from shark, skate, and ray cells and tissues that have the potential to inhibit disease processes in humans." Yun et al. (2005) recently reported that in early marine ecosystems where ocean-surface vegetation, which concentrates iodine for its antimicrobial and antioxidant properties, formed the basis for the food chain accumulation of antimicrobials that also exhibit antioxidant properties. These authors reported that “iodine may have emerged as a substrate for production of thyroid hormone in prehistoric ecosystems because the former represented a reliable proxy for ecologic potential that enabled the latter to modulate growth, reproduction, metabolic rate, and lifespan.” In the amphibians, environmental iodine is the essential metamorphosis factor and has an important role in the spectacular apoptosis on the cells of larval gills, tail, fins and gastro-intestine, and in adapting and transforming of aquatic animal (tadpole) to a “more developed” terrestrial animal (frog). In fact, programmed cell death, with nuclear changes and removal by phagocytic macrophages, occurs in a variety of organs, during amphibian metamorphosis, that is under the control of iodinated thyroid hormone. Indeed, because of the massive cell death that occurs during a short period, amphibian organs serve as an ideal model system for the study of mechanisms underlying programmed cell death (Ishizuya-Oka et al. 2003; Ikuzawa et al, 2005).

Fig. 2. In amphibian metamorphosis iodides and thyroxine have an important role in the spectacular apoptotic action on the cells of gills, tail and fins, and in adapting and transforming an aquatic animal (tadpole) into a “more developed” terrestrial animal (frog).

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15 To adapt to terrestrial carnivorous life, the amphibian gastro-intestine dramatically undergoes remodelling from larval to adult form during a short period of time. At the cellular level, the primary (larval) epithelium undergoes apoptosis, whereas a small number of undifferentiated cells appear, replace the larval epithelium by active proliferation, and newly form the secondary (adult) epithelium. In the intestine, the adult epithelium acquires the cell renewal system along the troughcrest axis of newly formed folds analogous to the mammalian crypt-villus axis. Thyroid hormone induces apoptosis of larval cells and differentiation of pepsinogen-producing cells in the stomach of Xenopus laevis in vitro (Ishizuya-Oka et al., 2003). Ikuzawa et al. (2005) reported that expression of CCAAT/enhancer binding protein delta (C/EBP delta) is closely associated with degeneration of surface mucous cells of larval stomach during the thyroid hormone-induced metamorphosis of amphibian Xenopus laevis, suggesting a possibility that Xenopus C/EBP delta plays a role in apoptotic cell death of larval-type epithelium during the stomach remodelling induced by thyroxine. Upadhyay et al. (2002) reported that excess iodine has been observed to induce also apoptosis in thyrocytes and mammary cells. According to Upadhyay, mitochondria are important in iodination of different proteins; mitochondria are the central executioner of apoptosis and therefore may play an important role in carcinogenesis. Mitochondrial proteins from breast tissue are iodinated. Organification of iodine to proteins requires oxidative enzymes, H2O2 generating system and proteins in the vicinity, conditions favourable for the iodination of proteins, which exist in mitochondria under normal circumstances. Upadhyay observed that mitochondria isolated from the tumour (TT) and extra-tumoral tissue (ET) of human breast display significant uptake of iodine. Mitochondrial proteins were observed to be predominantly iodinated in ET but not in TT mitochondria. Treatment with iodine showed an increase in mitochondrial permeability transition of TT and decrease in ET. Iodine induced released factors other than cytochrome c from tumour mitochondria initiate apoptosis in vitro.

Iodine in Terrestrial Organisms When about 400-300 Mya some living plants and animals began to transfer from the sea to rivers and land, environmental iodine deficiency was a challenge to the evolution of terrestrial life (Venturi, 2000). In marine-fishes, plants and animals the terrestrial diet became deficient in many essential marine trace elements, including iodine, selenium etc. Terrestrial plants, in replacement of marine antioxidants, slowly optimized the production of other endogenous antioxidants such as 15

16 ascorbic acid, polyfenols, carotenoids, flavonoids, tocoferols etc., some of which became essential “vitamins” in the diet of terrestrial animals (vitamins C, A, E, etc.). According to Coic and Coppenet (1990) and Lamand (1991), iodine and selenium became no longer necessary to plant life. In fact, photosynthetic terrestrial plants, producing into their cells poisonous oxygen, required protective substances and developed many and phylogenetically “new” endogenous antioxidants. Differently from plants, some primitive chordates, about 500 Mya, began to use also the “new” thyroidal follicles, as reservoir for iodine, and to use the thyroxine in order to transport antioxidant iodide and triiodothyronine into the peripherical cells. Triiodothyronine (T3), the biologically active form of thyroid hormone in vertebrates, became active in the metamorphosis and thermogenesis for a better adaptation to terrestrial fresh-waters, atmosphere, gravity, temperature and diet. The antioxidant action of iodides has also been described in brain cells of rats (Katamine, ’85) and in the therapy of some human chronic diseases of cardiovascular and articular systems (Winkler et al., 2000). Liu (2000.a, 2000.b) showed that ROS and lipid peroxidations increase in I-deficient rats and children. Recently, Aceves et al. (2005) and Cann (2006) suggested that iodides seem to have preventive effects in breast cancer and in cardiovascular diseases. Altorjay et al. (2007) reported that NIS expression is markedly decreased or absent in case of intestinalization or malignant transformation of the gastric mucosa, and suggested that NIS may prove to be a significant tumor marker in the diagnosis and prognosis of gastric malignancies and also precancerous lesions such as Barrett mucosa. Gelb et al. (1962) showed iodine binding by proteins in gastric mucus. Gołkowski et al. (2007) reported that iodine-prophylaxis is the protective factor against stomach cancer in iodine deficient areas. In 1883, Kocher observed that atherosclerosis, a suspected ROS-caused

disease, frequently appeared following thyroid extirpation and suggested that hypothyroidism may be causally associated with atherosclerosis. In 1930-50’s, potassium iodide has long been used empirically in patients with arteriosclerosis and cardiovascular diseases by European physicians (Cann, 2006), and Turner (1933a, b) reported the efficacy of iodine and desiccated thyroid in preventing the development of atherosclerosis in rabbits. Extrathyroidal or peripheral thyroid hormone metabolism is mediated by deiodinases [type 1 deiodinase (D1), type 2 deiodinase (D2) and type 3 deiodinase (D3) ]. D1 and D2 catalyse the conversion of T4 into T3, whereas D3 catalyses the inactivation of T4 into rT3 and of T3 into 3,3-T2 and in this way D3 delivers into peripheral cells one or two atoms of iodide per molecule of T4 without hormonal action. D3 protein is also expressed by granulocytes and monocarboxylate transporter 8 (MCT8), a novel very active and specific thyroid hormone transporter is also present at the site of inflammation. Thyroxine, reverse-T3 and iodothyronines are important as antioxidants and inhibitors of lipid peroxidation, more effective than vitamin E, glutathione and ascorbic acid (Cash et al. 1966; Ware and Wishner 16

17 1968; Tseng and Latham, ’84; Oziol et al. 2001; Berking et al. 2005). Thyroid cells phylogenetically derived from primitive gastroenteric cells, which were able to form iodo-tyrosines and, during evolution, migrated and specialized in uptake and storage iodine-compounds in the new follicular structure. Moreover the new terrestrial diet harboured plant iodide-transport inhibitors such as thiocyanates, cyanates, nitrates and some glycosides ( Wolff, ’64; Brown-Grant, ’61). Many plant substances that inhibit iodide-transport (as metabolic inhibitors, transport inhibitors or competitive anions) seem to have antiparasitic activity ( Wolff, ’64). The thyroid gland, with a progressively more developed morphology appeared and was improved from primitive Chordata to more recent marine and fresh-water fishes, Amphibia, reptiles, birds, and finally mammals (Venturi et al., 2000a). Venturi et al. (2000) reported that, contrary to amphibian metamorphosis, in the mammals the thyroidectomy and hypothyroidism might be considered like a sort of phylogenetical and metabolical regression to a former stage of reptilian life. In fact, many disorders, similar to reptilian features, such as a dry, hairless, scaly, cold skin and a general slowdown of metabolism, digestion, heart rate, nervous reflexes with lethargic cerebration, hyperuricemia, and hypothermia seem to afflict hypothyroid humans. The new hormonal action was made possible by the formation of T3-receptors (TH-Rs) in the cells of vertebrates. First, about 500 Mya, in primitive marine chordates, the primitive TH-Rs with a metamorphosing action appeared and then, about 250-350 Mya, in the birds and mammalians, others more recent TH-Rs with metabolic and thermogenetic actions were formed. TH-R genes are indeed c-erbA oncogenes , which have been implicated as tumour suppressor genes of non-thyroidal cancers and are altered in some human gastric and mammary cancer (Wang et al., 2002; Li et al. 2002).

Role of Iodide in Animal Cells In humans, the total amount of iodine is about 25-50 mg and about 50-70 % of total iodine is nonhormonal and it is concentrated, via NIS, in extrathyroidal tissues. In 1996, cloning and molecular characterization of the human NIS have been performed (Dai et al, ’96; Smanik, ’96). In the thyroid cells active iodide transport is facilitated by three transporters, NIS, Pendrin, and the recently described the apical iodide transporter (AIT). Expression of all three transporters appears in extrathyroidal tissues (Burbridge, 2005; Rodriguez et al., 2002). Iodine is present, in different concentration, in every organ and tissue of the human body, not just the thyroid gland. So far the list of these iodide-concentrating cells includes: white blood cells, salivary and lacrimal glands, ciliary body of the eye, renal cortex, the pancreas, the liver, gastric, small and large intestinal mucosa, 17

18 nasopharynx, choroid plexus, skin, adrenal cortex, mammary gland, placenta, uterus and ovary (Brown-Grant, ’61).

Fig. 3. Sequence of I-123 total-body scintiscans of a woman after intravenous injection of I-123 (half-life: 13 hours); (from left) respectively at 30 minutes, and at 6, 20 and 48 hours. It is evident the highest and rapid concentration of radio-iodide (in white) in gastric mucosa of the stomach, salivary glands and oral mucosa. In gastric mucosa of the stomach, 131-I (half-life: 8 days) persists in scintiscans for more than 72 hours. In the thyroid I-concentration is more progressive, as in a reservoir [from 1% (after 30 minutes) to 5.8 % (after 48 hours) of the total injected dose]. Mammary gland iodide-concentration is here not evident because this woman was not pregnant or lactating. It is evident a high excretion of radioiodide in urina.

We hypothesize that both these actions of iodine may still have an important place in the cells of modern vertebrates (Venturi and Venturi, ’99; Venturi et al., 2000a, b; Venturi et al., 2003 ). In fact, Evans et al. (1966) reported that 5 mg of potassium iodide (daily injected) acts directly as 0.25 µg of L-thyroxine (indirectly), in recovering the impaired functions of many organs of thyroidectomized rats. This suggests that while iodides are always necessary, the thyroid hormones (via indirect action) and all known TH-receptors do not seem to be essential to living organisms, as reported by Goethe (1999) and Wolff (1964). Dobson (1998) suggested that Neanderthal man suffered I-deficiency disorders caused by inland environment or by a genetic difference of his 18

19 thyroid compared to the thyroid of the modern Homo Sapiens. I-deficient humans, like endemic cretins, suffer physical, neurological, mental, immune and reproductive diseases. Iodine has favoured the evolution of the nervous system for a better adaptation to terrestrial environment as recently reported by Cunnane (2005), who suggested that “iodine is the primary brain selective nutrient in human brain evolution.” Cordain et al. (2005) recently reported that the profound changes in diet, that began with the introduction of agriculture and animal husbandry approximately 10,000 years ago, occurred too recently on an evolutionary time scale for the human genome to adjust. In conjunction with this discordance between our ancient, genetically determined biology of hunter-gatherers and fisher-gatherers human societies and the nutritional patterns of contemporary Western populations, many of the so-called diseases of civilization have emerged. Cordain suggests that iodine deficiency was probably one, among other dietary variations, introduced during the Neolithic and Industrial Periods, which have altered crucial nutritional characteristics of ancestral human diets (simultaneously fibre and polyunsaturated fatty acid contents). The evolutionary collision of our ancient genome with the nutritional qualities of recently introduced foods may underlie many of the chronic diseases of Western civilization. Recently was reported that TascoForage, an (iodine-rich) extract from the brown seaweed Ascophyllum nodosum, has increased antioxidant activity and immune system in both plants and in grazing animals (Saker et al., 2001, 2004; Montgomery et al, 2001; Allen et al., 2001; Fike at al., 2001). Iodine at physiological doses possibly supports anticarcinogenic defence of the organism. Some data evidenced that iodine can prevent breast and gastric cancer in humans (Eskin, ’70, ’77; Venturi, 2000b, 2001; Funahashi et al., ’96, 2001; Smyth, 2003a, b; Kessler, 2004; Szybinski et al., 2004; Abnet et al., 2006 ). Aceves et al. (2005) reported that “Iodine is a gatekeeper of mammary glad integrity” and proposes that an iodine supplement should be considered as an adjuvant in breast cancer therapy. In the same Ideficient territories, human and animal pathologies by I-deficiency frequently coexist in the mammals (in particular herbivores) and in reptiles, amphibians and fresh-water fishes too. The evolutionary adaptation of terrestrial vertebrates to environmental iodine deficiency is still not finished yet (Cabello et al., 2003), and most humans and terrestrial animals still need a dietary iodine supplementation (Food and Nutrition Board, USA, 2001). According to current W.H.O. statistics more than 3 billion people in the world live nowadays in I-deficient countries. In the analysis of “National Health and Nutrition Examination Surveys” data of moderate to severe iodine deficiency is present now in a significant proportion (11.7%) of the U.S.A. population, with a clear increasing trend over the past 20 years, caused by reduced iodized table salt usage (Hollowell et al., ‘98 ).

19

20 In conclusion, we suggest that some primitive marine antioxidants, as iodide and selenium, are apoptosis-inductors and seem to have an anti-tumour activity. Studies of molecular evolution of primitive antioxidants might give the basis for further research of “new” protective substances against many chronic and degenerative diseases.

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