Implications and Practical Applications of the Chemical Speciation of Iodine in the Biological Context

: Iodine is a highly reactive element with a single natural and stable isotopic form ( 127 I). In the biosphere, it is one of the 30 essential elements for life, and its chemical speciation deﬁnes its availability and biological activities. The most relevant chemical species are iodate (IO 3 − ) and iodide (I − ) as the major sources of iodine, with molecular iodine (I 2 ) and hypoiodous acid (HIO) as the most reactive species, and thyroid hormones (THs) as the representative organic compounds. In human biology, THs are master regulators of metabolism, while inorganic species serve for the iodination of organic molecules and contribute to the innate immune system and the antioxidant cellular defense. Additionally, I − , I 2 , δ -lactone (6-IL), and α -iodohexadecanal ( α -IHDA) have shown therapeutic potential in counteracting oxidative stress, cancer, and inﬂammation. Both inorganic and organic species have applications in the health science industry, from the manufacturing of disinfection and wound care products to supplements, medicines, and contrast media for radiography. Even after nuclear disasters, intake of high doses of iodine prevents the accumulation of radioactive iodine in the body. Conversely, the controlled production of iodine radioisotopes such as 123 I, 124 I, 125 I, and 131 I is exploited in nuclear medicine for radiotherapy and diagnostics. of proposed molecular mechanisms of iodine I as a cofactor in PO reactions I α -IHDA, 6-IL); (2) a direct scavenger of ROS produced by mitochondria (OH • radical O 2 − ) I 2 HIO and HI); activation of the Nrf2–ARE pathway; inhibition of pro-oxidant and proinﬂammatory enzymes Cox2 by Author Contributions: Conceptualization, A.N.E.-V., L.M.F.-Y. and F.C.R.-C.; investigation, A.N.E.-V.; writing—original draft preparation, A.N.E.-V.; writing—review and editing, A.N.E.-V., L.M.F.-Y. and F.C.R.-C..; visualization, A.N.E.-V. and L.M.F.-Y.; supervision, L.M.F.-Y. CXC DIT 3,5-Diiodotyrosine, EGF Epidermal Growth Eh Redox potential, EPO Eosinophil peroxidase, ERK1/2 Extracellular signal-Regulated 1/2, G-POX Guaiacol peroxidase, GSH Reduced glutathione, GSSG Oxidized glutathione, H 2 O 2 H 2 OI + Iodine cation, HDL-C High-density lipoprotein cholesterol, HI HIO Hypoiodous HIO 3 Iodic acid, HO-1 oxygenase I Iodine element, I − Iodide, I 2 molecular iodine, I 2 O 5 Iodine pentoxide, I 3 − Triiodide, I 5 − Pentaiodide, I 62 − Hexaiodide, ID Iodothyronine deiodinase, IDD Iodine Deﬁciency γ Interferon-gamma, IGT Impaired glucose tolerance, IL Interleukin, IO 3 − Iodate ion, IP3 Inositol 1,4,5-triphosphate, IYD Iodotyrosine deiodinases, Keap1 Kelch-like ECH-associated KI Potassium iodide, KIO 3 iodate, LDL-C Low-density lipoprotein cholesterol, Lipoxygenase, LPO Na 2 NADPH NF- κ B Nuclear kappa-light-chain-enhancer of activated B, NH 2 C Chloramine, NIS Na + /I − symporter, NMNH Reduced dihydronicotinamide mononucleotide, NO–cGMP Nitric oxide–cyclic guanosine monophosphate pathway, NOS nitric oxide synthase, NR Nuclear Receptors, Nrf2 Nuclear factor erythroid 2-related factor 2, O 2 − Superoxides, O 3 Ozone, OATP Organic Anion-Transporting Polypeptide, OH • Hydroxyl radical, O I − Hypoiodite ion, PCNA Proliferating Cell Nuclear Antigen, PCNA Proliferating Cell Nuclear Antigen, PDD Hydrogen peroxide-dependent diffusion, PECT Positron Emission Tomography, PI3K Phosphatidylinositol 3-OH Kinase pathway, PPAR α Peroxisome Proliferator-Activated Receptor type alpha, PPAR γ Peroxisome Proliferator-Activated Receptor type gamma, PVAI Polyvinyl Alcohol Iodine, PVP-I Povidone-Iodine, RIA Radioimmunoassay, rT3 Reverse T3 or 3 (cid:48) ,5 (cid:48) ,3 triiodothyronine, SMVT Sodium-dependent Multivitamin Transporter, SOD Superoxide dismutase, SPECT Single-Photon Emission Computed Tomography, T3 3 (cid:48) ,5,3-Triiodothyronine, T4 Thyroxine, TAAR Trace amine-associated receptor, TAC Total Antioxidant Capacity, TAMs Thyronamines, Tetrac 3,3 (cid:48) ,5,5 (cid:48) -Tetraiodothyroacetic acid, TG Thyroglobulin, TGF Transforming Growth Factor, THs Thyroid Hormones, TPO Thyroid Peroxidase, TR Thyroid hormone receptors, TREs Response Elements, TRH Thyrotropin-Releasing Hormone, Triac 3,3 (cid:48) ,5-triiodothyroacetic acid, TRs Thyroid hormone


Introduction
Life originated in the oceans, where iodine is available in high amounts, and it is believed that, since then, it has had an ancient role in the biology of emergent organisms [1]. This element is essential in the metabolism of countless living beings from the three major domains of life [1]. Yet, depending on the chemical species and their amount, iodine acts as a nutrient showing protective roles or as a cellular stressor. For instance, catalytic inorganic iodine species inhibit microorganisms, attacking structural components such as nucleic acids, proteins, and lipids in a dose-dependent manner [2]. Contrary, in appropriate amounts, eukaryotic cells have taken advantage of the reactivity of some inorganic species to synthesize new compounds, counteract cellular oxidative stress, and modulate cell signaling pathways [3].
Despite the vast information around iodine, we consider that most recent studies have not entirely regarded critical aspects of its chemistry and stability when discussing the novel roles of this element in biological systems. This review compiles the multidisciplinary evidence of iodine, considering the characteristics of its biologically active forms to better understand the functions, modes of action, and applications. For that, we first describe the basic features of the element, such as the history, physicochemical properties, and geological distribution. Then, we list the most relevant inorganic and organic iodine species and state the fundaments of the chemical speciation, especially under biological conditions. With those bases, we further expose and discuss the known roles and activities of the main iodine species, considering evolutive aspects. Lastly, we connect the presented information

Main Inorganic Forms
Dominant iodine species in the solid state are potassium iodate (KIO 3 ) and potassium iodide (KI), whereas IO 3 − and I − ions lead to aqueous systems [15]. I 2 and CH 3 I are the most abundant species in the atmosphere, produced by biotic and abiotic processes [16]. Atmospheric photochemistry promotes the speciation of gaseous iodine species (e.g., HI, IO − , HIO, IONO 2 INO 2 , and IO 2 ) [17,18]. Some of them (mainly I 2 and HIO), together with other halogen compounds, are responsible for ozone (O 3 ) depletion in the lower troposphere when interacting with hydrogen-oxygen species and nitrogen oxides [19].
Iodate is the most thermodynamically stable of all iodine species and has no oxidative action in the basic and neutral pH range; it only achieves some reactivity at very low pH, such as HIO3 (pKa = 0.77) [24]. The main sources are calcium iodate (Ca(IO3)2), potas- Iodine speciation in aqueous systems. Pourbaix diagram of iodine depicting species stability by pH and Eh [26]. Abbreviations: pH, potential of hydrogen; Eh, redox potential (measured in volts).
Iodine in iodides I 3 − and I 5 − have oxidation states of -1, in contrast to an oxidation state of 0 in I 2 . Higher values exist when iodine combines with oxygen, such as in HIO, iodite (IO 2 − ), iodate (IO 3 − ), and periodate (IO 4 − or IO 6 5− ) having oxidation states of +1, +3, +5, and +7, respectively [10]. The oxidation state refers to the electronic configuration of an atom in a chemical species and represents the number of electrons gained (-) or lost (+) [10].
Iodate is the most thermodynamically stable of all iodine species and has no oxidative action in the basic and neutral pH range; it only achieves some reactivity at very low pH, such as HIO 3 (pKa = 0.77) [24]. The main sources are calcium iodate (Ca(IO 3 ) 2 ), potassium iodate (KIO 3 ), and potassium biiodate (KH(IO 3 ) 2 ), some of them with better water solubility than iodides (e.g., HIO 3 = 308 g/100 g H 2 O at 25 • C vs. KI = 148 g/100 g H 2 O at 25 • C) [31]. IO 3 − represents a major source of iodine for living beings and is well absorbed in several organisms. Hence, it needs to be transformed into a different species to exhibit an active biological role, as described in sections below [24].
Thyroid peroxidase (TPO) is the enzyme responsible for synthesizing THs using thyroglobulin (TG) as a substrate. In fact, TG is the main iodinated compound in the thyroid gland [42][43][44]. TPO is also responsible for the iodination of lipids (iodolipids) as α-IHDA and 6-IL. Noticeably, 6-IL was recently related to antioxidant, anti-inflammatory, and anticarcinogenic activities (see   [45,46]. Other peroxidase enzymes such as lactoperoxidase (LPO) are responsible for the synthesis of iodolactones (e.g., δlactone of 6-iodo-4-hydroxy-eicosa-8,11,14-trienoic acid, and ε-lactone of 5-iodo-4-hydroxy-7-docosapentaenoic acid) using docosahexaenoic acid (DHA) as a substrate [32]. TPO and LPO belong to the peroxidase/cyclooxygenase superfamily, a class of oxidoreductase enzymes broadly distributed in the tree of life. Plants, fungi, bacteria, and animals use them to maintain oxidative status and synthesize new compounds or even as part of the innate host defense [47].
Some organisms unable to produce their own iodinated organic compounds depend on symbiotic relationships to supply them. Sea urchins, for example, need THs to metamorphose from larva to adult rudiment but obtain them from algae feeding [48]. Symbiosis also impacts the iodinated compounds profile of microorganisms. The enteric bacterium Escherichia coli cannot use I − , I 2 , MIT, or DIT, but takes T4 and T3 to iodinate proteins [49,50]. Even viruses influence the production of iodinated organic compounds of their hosts. Recently, it was observed that a cyanophage (cyanobacteria virus) encodes for a halogenase (an enzyme that replaces hydrogen atoms with halogen atoms in a molecule) with the potential of iodinating organic molecules of its host [51]. Moreover, the presence of viruses in Emiliania huxleyi algae changes its metabolic profile. After viral infections, algal blooms (excessive growth of marine photosynthetic organisms) discharge a large amount of organic matter containing chloro and iodinated metabolites [52]. More examples of iodinated organic compounds can be consulted in [32].

Antimicrobial Action
Iodine shows a broad antibacterial spectrum against several microorganisms, including resistant forms (e.g., spores, cysts, and biofilms) [53][54][55]. Of all inorganic iodine species with antimicrobial activity, I 2 is the most abundant and the leading antimicrobial agent in iodine-based disinfectants [24]. Other iodine species recognized as antimicrobials are HIO and H 2 OI + ; however, as mentioned, they are restricted to extreme pH ranges and by the amount of I − [27,28]. Within solvents like alcohol, the solvated forms I 2 H 2 O or I 2 ROH are proposed as the antimicrobial species [2].
Contrary to antibiotics or antifungals, microorganisms do not develop antimicrobial resistance to iodine, probably because of its several simultaneous mechanisms of action. In bacteria, iodine inhibits glycolysis, respiration, glucose transport, and DNA and RNA synthesis, and it compromises the integrity of the plasma membrane and cell wall. Antimicrobial iodine species are also responsible for protein (and potentially DNA) denaturalization in bacteria and viruses and damage viral envelopes [27,56,57].
The molecular modes of action described for I 2 and HIO are oxidation of nucleotides (adenine, cytosine, and guanine) and aromatic rings of amino acids (mainly Tyr) [57], interfering with the synthesis and folding of proteins via iodination of amino (-NH) (Reaction (13) and (14)) and sulfhydryl (-SH) groups (Reactions (15)-(17)) [28,30,57], or disrupting disulfide bonds (R-S-S-R) between cysteines (Reactions (10)-(12)) [58]. They also have a great affinity to unsaturated fatty acids, attacking the carbon-carbon double bonds (Reaction (18)) [30,59]. Cation H 2 OI + is also responsible for attacking the -SH groups of some amino acids (Reactions (19) and (20)) and iodination of tyrosine and histidine residues (Reactions (21)-(23)) [56,57]. The following reactions have been proposed for antimicrobial iodine species (most of them as chemical skeletal equations): Tyr TyrI His Eukaryotes take advantage of the reactivity of some iodine species to counteract infections, among other biological activities (see ). In mammals, antimicrobial iodine species can be produced as byproducts of peroxidases. Depending on the pH and I − amounts, LPO can produce HIO and I 2 by reducing hydrogen peroxide (H 2 O 2 ) to H 2 O, using I − as the electron donor (Reaction (24)) [60,61]. LPO is secreted in mucosal surfaces or exocrine secretions such as saliva, milk, or tears and is essential for the innate immune defense system [28,47]. The sole action of LPO is bacteriostatic; however, in conjunction with I − , it becomes bactericidal and antiviral [62][63][64].

Nutrition and Human Health
Together with vitamin A, iron, zinc, folate, and vitamin B-12, iodine is considered an essential micronutrient for humans and an important marker of nutritional public health status [65]. An optimal iodine status (sufficiency) can be maintained with an appropriate diet and supplements (see Section 7.2); however, inadequate iodine ingestion drives severe health consequences in the long term, known as iodine deficiency disorders (IDD). The most common are hypothyroidism, endemic goiter (enlargement of the thyroid), and thyroid nodules. Pregnant women and fetuses are the most susceptible to IDD, inducing abortions, perinatal mortality, and congenital anomalies [66][67][68]. Other expected health consequences are cretinism and delayed physical development in infants and children. Cognitive impairment and increased susceptibility to nuclear radiation are seen at all ages under iodine deficiency [67,69].
Thyroid disorders in the population and exposure to iodine are correlated in a Ushaped curve, as depicted in Figure 3A, where the higher risk comes with the chronic suboptimal intake of iodine (iodine deficiency) [67,70]. Excess exposure to iodine is less common but can be dangerous, especially in iodine-deficient populations. Excessive intake is frequently associated with high consumption of iodized salt and diet [71,72], but other sources came from medications such as amiodarone (200-400 mg/day) [73], radiologic procedures (one dose of >350 mg) [74], and povidone/iodine (PVP/I)-based antiseptics (<30 mg/day) [75]. Excess iodine increases the risk of developing transient or permanent thyroid dysfunction (iodine-induced hyperthyroidism or hypothyroidism), thyroid autoimmune diseases, metabolic disorders, metabolic syndrome, hypertension, prediabetes, diabetes mellitus, impaired glucose tolerance, central obesity, and dyslipidemia ( Figure 3B-F) [70,[76][77][78]. Vulnerable groups such as the elderly, fetuses, neonates, and people already having an IDD are more susceptible to iodine-induced hyperthyroidism or hypothyroidism. In the best scenario, hypothyroidism only lasts 2-10 days (escape from acute Wolff-Chaikoff effect), but might be lethal in fetuses or neonates. Iodine-induced hyperthyroidism or Jod-Basedow syndrome has greater incidence in adults and can be related to cardiac complications [39,78].
People with normal thyroid functioning (euthyroid) tolerate excessive iodine well. In the Japanese population, the regular consumption of iodine-rich foods correlates with a higher life span, as well as fewer infant deaths, cardiac diseases, and breast and prostate cancer incidences, than in the US, whose iodine intake is typically much lower. On average, people consume 1-3 mg/day of iodine in Japan, where one of the primary sources is seaweed-based meals such as nori (Porphyra), wakame (Undaria), and kombu (Laminaria) [80]. Interestingly, these health markers are not attributable to genetics; Japanese emigrants increased their risk of mammary cancer from 21.1 to 49.4 per 100,000 population and from 8.4 to 32.2 of prostate cancer per 100,000 just by changing diet [81,82]. Currently, the incidence of both types of cancer in Japan is increasing, probably because of the occidentalization of diet. Prostate cancer had the highest frequency in 2020 (close to 96,000), and breast cancer new cases were over 50,000 in 2018, compared to 2003 [83]. Nevertheless, thyroid cancer represents the rarest cause of death from cancer for Japanese (600 deaths for men in 2018 and 1200 for women in 2019) [83].
Iodine sufficiency areas have a lower risk of developing IDD; however, they are not exempt from Graves' disease (with~80% incidence in iodine-sufficient populations), nodular thyroid disease, and thyroiditis [84]. Respecting thyroid cancer (the most common endocrine cancer), some authors conclude that more than the recommended iodine ingestion could increase papillary and thyroid cancer [38,72], but others observed a lower incidence [85]. A more robust meta-analysis of humans and animals found iodine deficiency as a promotor risk factor for follicular, and possibly anaplastic, thyroid cancers [86].  [79], Academic Press, 2009). Relationship between UIC (µg/L) and the prevalence (%) of (B) metabolic disorders and MetS, (C) prediabetes, diabetes, and IGT, (D) hypertension and central obesity, (E) hypertriglyceridemia and hypercholesterolemia, and (F) HDL-C and LDL-C (data from [70], Mary Ann Liebert, Inc., publishers). Abbreviations: HDL-C, high-density lipoprotein cholesterol; IGT, impaired glucose tolerance; IDD, iodine deficiency disorders; LDL-C, low-density lipoprotein cholesterol; MetS, metabolic syndrome; UIE, urinary iodine excretion; UIC, urinary iodine concentration.
People with normal thyroid functioning (euthyroid) tolerate excessive iodine well. In the Japanese population, the regular consumption of iodine-rich foods correlates with a higher life span, as well as fewer infant deaths, cardiac diseases, and breast and prostate cancer incidences, than in the US, whose iodine intake is typically much lower. On average, people consume 1-3 mg/day of iodine in Japan, where one of the primary sources is seaweed-based meals such as nori (Porphyra), wakame (Undaria), and kombu (Laminaria) [80]. Interestingly, these health markers are not attributable to genetics; Japanese emigrants increased their risk of mammary cancer from 21.1 to 49.4 per 100,000 population and from 8.4 to 32.2 of prostate cancer per 100,000 just by changing diet [81,82]. Currently, the incidence of both types of cancer in Japan is increasing, probably because of the occidentalization of diet. Prostate cancer had the highest frequency in 2020 (close to 96,000), and breast cancer new cases were over 50,000 in 2018, compared to 2003 [83]. Nevertheless, thyroid cancer represents the rarest cause of death from cancer for Japanese (600 deaths for men in 2018 and 1200 for women in 2019) [83].

TH Synthesis and Metabolism
From the anthropocentric point of view, the leading role of iodine is TH production in the thyroid gland, also known as organification. THs are iodotyrosines that can be produced by peroxidase enzymes (such as LPO and TPO) or via nonenzymatic reactions of iodine oxidative species (such as I 2 ) with Tyr residues (Reaction (25)) [47,87]. This section focuses on the TH synthesis carried out in vertebrate animals. The occurrence of THs in different organisms and their functions is discussed next.
The regulation of the thyroid function is a circuit known as the thyroid axis, where, in normal conditions, TH synthesis and secretion increase in response to the thyroidstimulating hormone (TSH) produced by the anterior pituitary gland [88]. TSH is stimulated in turn by the thyrotropin-releasing hormone (TRH), released by the hypothalamus [89]. Excess circulating T4 or T3 inhibits the hypothalamus from secreting more TRH, resulting in a decrement in TSH and, hence, in TH production [90].
As an initial step in TH synthesis, iodine must be absorbed into the body. Iodide is not diffused but is actively imported by transmembranal transporters into the bloodstream [91]. Most intake of iodine (>90%) is absorbed in the digestive tract, mainly in the small intestine by the Na + /I − symporter (NIS) [92]. Depending on the iodine status (sufficiency, deficiency, or excess), the thyroid imports <10% up to 80% of the circulating iodine. Unused I − is mainly excreted in the urine (~90%), depending on the glomerular filtration rate, with a lesser proportion in feces [39,65].
TH synthesis occurs in the thyroid follicles, composed of a colloid (follicular lumen) enclosed by follicular cells and surrounded in the interfollicular space by blood vessels and parafollicular cells [93]. In the basement membrane of follicular cells, I − is imported by the Na + /I − symporter (NIS) and moves at the apical membrane into the follicular lumen through different transporters such as the anion exchanger Pendrin, the calcium-dependent Cl − channel Anoctamin 1 (ANO1), and possibly others such as cystic fibrosis transmembrane conductance regulator (CFTR), Cl − /H + antiporter (ClC5), Cl − /H+ exchanger (ClC-3), and the sodium-dependent multivitamin transporter (SMVT) [94][95][96][97]. Once in the lumen, I − needs to be oxidated into a more reactive form by the TPO. The specific iodine species formed in this step has not been determined, but the likely candidates are I 2 , iodinium (I + ), iodine free radical (I • ), and hypoiodite (IO − ) [98].
TPO, coupled to the apical membrane of the follicles, is responsible for iodination and coupling of Tyr residues using I − , H 2 O 2 , and thyroglobulin (TG) as the substrate (a large glycoprotein of~330 kDa) [42]. H 2 O 2 is produced by a complex formed by the dual oxidase 2 (DUOX2, formerly THOX2) and dual oxidase maturation factor 2 (DUOXA2), also coupled to the apical membrane [99]. The sequence followed in TH production is incorporating one iodine atom into Tyr residues at position 3 of the ring to produce MIT (Reaction (26)) and then at position 5 to form DIT (Reaction (27)). Subsequently, T3 is constructed by an oxidative coupling reaction between one DIT and one MIT (Reaction (28)), still incorporated into the TG. At the same time, T4 is formed by coupling two adjacent DITs (Reaction (29)). At the end of the process, only 37 of the~70 Tyr residues in the TG are known iodination targets [42,43]. To release THs, iodinated TG is transported back into follicular cells by endocytosis and hydrolyzed by proteolysis [42,90]. Most MITs and DITs produced are kept in the follicles and are eventually degraded by iodotyrosine deiodinase (IYD) enzymes into MIT and Tyr, respectively (Reactions (30) and (31)), releasing I − which is reused and recycled [100]. However, T3 and T4 are transported into the bloodstream, mostly bound to plasma proteins. The most common are thyroxine-binding globulin (carrying up to 80% of bound T3 and~65% of bound T4), transthyretin (transporting~9% of T3 and~15% of T4), and albumin (with~11% of T3 and~20% of T4). A lower percentage of THs circulate as free hormones (approximately 0.2% of T3 and 0.05% of T4) [40].
Tyr residue 2DIT In the blood, T4 has the highest concentration (54-174 µg/dL) and most prolonged half-life (~7 days), but T3 (half-life < 12 h) has a higher proportion in organs, especially the kidney, liver, small and large intestine, and central nervous system [40]. Just a fraction of T3 (21.8%) is synthesized and released in the same fashion as T4; most of it is generated by the deiodination of T4 out of the thyroid by iodothyronine deiodinases (ID) [90,101]. Iodine removal serves to activate or inactivate THs. D1 and D2 are the two kinds of ID enzymes considered as activators by converting T4 into T3 (Reaction (32)) [90,100]. D3 transforms T4 into rT3 (Reaction (33)), an inactive TH produced to maintain hormone homeostasis [102].
Deiodination is considered the most important process in the peripheral metabolism of THs, followed by conjugation, formation of analogs (via deamination and decarboxylation), and oxidation [103]. Conjugation of THs with sulfate or glucuronic acid (via microsomal glucuronyl or cytoplasmic phenol transferases) inactivates and increases the solubility of hormones, accelerating their deiodination and clearance by biliary excretion [104].
The gut microbiota also significantly contributes to TH metabolism, favoring the cycling of I − and THs [101,102]. On the one hand, enteric bacteria are proposed as TH storage because of their ability to absorb and retain T3 and T4 [49,105]; on the other hand, the gut microbiota releases sulfatase and glucuronidase enzymes favoring the enterohepatic circulation (secretion of conjugated THs in the bile to the small intestine and back to the liver after deconjugation) [106]. Moreover, gut bacteria influence the absorption of nutrients that support the normal thyroid metabolism, such as iron, selenium, and zinc [107]. The importance of the gut microbiome in thyroid function is more evident in autoimmune thyroid diseases, where an altered microbiota composition (dysbiosis) is linked to predisposition to Hashimoto's thyroiditis and Graves' disease. Furthermore, a reduction in or lack of Lactobacillaceae and Bifidobacteriaceae families in the gut has been related to autoimmune metaplastic atrophic gastritis [107,108].

Actions of THs
THs are needed in all stages of life, participating in numerous biological functions in vertebrates such as regulation of the brain function, hair growth, functioning of gonads (especially in females), thermogenesis (heat produced in the body), and response to the environment and stimuli. At the molecular level, THs regulate essential cellular processes such as development, differentiation, growth, maturation, and metabolism via different mechanisms, as described below [3,40].
Excluding activation/inactivation by deiodination or conjugation, the biological activities of THs are determined by the hormonal levels inside the cells (controlled by TH transporters), thyroid hormone receptors (TRs), and other elements such as corepressors and coactivators [109]. More than 20 TH transporters have been characterized, but those with the highest affinity are the monocarboxylate transporters MCT8 (transporting T4, T3, rT3, and 3,3 -T2) and MCT10 (transporting only T3 and T4), as well as the organic anion-transporting polypeptides, such as OATP1C1 (with affinity to T4, rT3, and T4 sulfate) and OATP-F (with affinity to T4 and rT3) [110,111].
Once inside, THs activate TRs in the nucleus and mitochondria. The complex of THs/TRs binds directly to DNA regions acting as transcription factors regulating the expression of several genes [112]. In humans, two genes encode for TRs (α and β), yielding, via alternative splicing, several TRα and TRβ isoforms depending on the tissues and developmental stage. For example, TRα1 is mainly expressed in the bones, heart, intestine, skeletal muscle, and central nervous system, while TRβ1 is better expressed in the liver, kidney, and inner ear [109,113,114]. Truncated TR forms are also produced by alternative translations of start codons (AUG) or internal promoters in introns. Their cellular location is linked to post-translational modifications such as palmitoylation, phosphorylation, acetylation, ubiquitination, and sumoylation [109].
Concerning the mode of action, THs typically follow two pathways: genomic (carried out in the nucleus) and nongenomic (faster and initiated in the cytoplasm, plasma membrane, mitochondria, and possibly others) [113]. A more recent classification proposes four types of mechanisms: (1) direct interaction of THs/TRs on the DNA (as the classic genomic mechanism), (2) indirect interaction of THs/TRs on the DNA, (3) TH/TR signaling without DNA binding, and (4) TH signaling independent of TRs [115]. The classical genomic mechanism involves the activation of TRs through a complex with T3 and other proteins in the nucleus. After that, the complex binds to regulatory DNA regions known as thyroid hormone response elements (TREs) to regulate their expression [40]. The indirect DNA binding mechanism (also considered a genomic pathway) implicates the interaction of TH and TRs with chromatin-associated proteins leading to chromatin accessibility and upregulation of gene expression [109].
Nongenomic mechanisms comprise alternative cellular compartments, novel interactors, and potentially less recognized THs. A type 2 mechanism (of the new classification) comprises the interaction of TRs with THs and kinases in the cytoplasm to activate cell signaling responses such as the phosphatidylinositol 3-OH kinase pathway (PI3K) (implicated in growth, proliferation, differentiation, etc.). Another nongenomic pathway (or type 3 mechanism in the new classification) starts with the activation of a structural protein in the plasma membrane, the integrin αvβ3 receptor (with binding domains for T4 and T3), which triggers PI3K and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, also a crucial regulator of the cell cycle, growth, and proliferation [113]. Similarly, a truncated TRα1 variant (p30) is found in the lipid rafts of the plasma membrane and activates the nitric oxide-cyclic guanosine monophosphate pathway (NO-cGMP) in response to T3 [109].
The mitochondrion is one of the main TH accumulators in the cell. Here, the oxidative phosphorylation system is highly influenced by different mechanisms involving truncated TRα1 variants (p28 in the inner membrane and p43 in the matrix). In the best-described pathway, T3 interacts with p43 on responsive elements of the mitochondrial DNA, regulating the transcription of respiratory genes (such as classic genomic mechanisms) [113,116]. The pathway that p28 follows is still unknown but shows a higher affinity to T3, and its expression affects mitochondrial physiology [117].
Previously, T3 was considered the biologically active hormone due to its higher affinity to most TRs [40]. However, the activities of T4 and other THs has been proven in different ways. For example, T4 but not T3 has the faculty to remodel the actin cytoskeleton of astrocytes and glial cells in the brain via nongenomic mechanisms [118]. Triac (antagonist to αvβ3) is better than T3 for increasing thermogenesis in brown adipocytes [119], and both Triac and Tetrac are preferable treatments for TH-resistant patients [120]. T1AM, the most potent antagonist of the trace amine-associated receptor 1 (TAAR1), can decrease heart output and body temperature [120,121]. T2 may prevent body weight gain, hepatic fat accumulation, and insulin resistance [120,122]. Moreover, T1AM and T2 modulate the mitochondrial oxidative phosphorylation system [116].

THs out of Vertebrates
Evolutionary, when marine organisms move to environments with less availability of iodine, they have to develop strategies to compensate for iodine deficiency. It has been postulated that our marine ancestors developed iodine-storage organs (endostyle and thyroid) when they moved from oceans to fresh waters and lands [1,3]. Lampreys, the most ancient vertebrates, have all the components of the thyroid system. These organisms transform from filter-feeder larvae (ammocoete stage) to juvenile and adult jawless fishes (many of them are fish parasites) by producing high levels of T3 and T4. The TH levels drastically fall during metamorphosis until the complete conversion into fish parasites, when lampreys move from freshwater to seawater [123]. Animals such as amphioxuses (a basal group of chordates) lack the thyroid but have the endostyle, a filter-feeding organ where iodine accumulation and TH synthesis occur ( Figure 4). Instead of T3 or T4, Triac is the active hormone in this model, binding with higher affinity to TRs and regulating the metamorphosis [124,125].
(many of them are fish parasites) by producing high levels of T3 and T4. The TH levels drastically fall during metamorphosis until the complete conversion into fish parasites, when lampreys move from freshwater to seawater [123]. Animals such as amphioxuses (a basal group of chordates) lack the thyroid but have the endostyle, a filter-feeding organ where iodine accumulation and TH synthesis occur (Figure 4). Instead of T3 or T4, Triac is the active hormone in this model, binding with higher affinity to TRs and regulating the metamorphosis [124,125]. Interestingly, many organisms along the three of life produce one or more components of the thyroid system ( Figure 4). Nuclear receptors (NRs) are found in all animals and can bind to several hormones (including THs); hence, they are considered ancestors of TRs [126]. TRs can be found in vertebrates and lineages of nonvertebrate deuterostomes Interestingly, many organisms along the three of life produce one or more components of the thyroid system ( Figure 4). Nuclear receptors (NRs) are found in all animals and can bind to several hormones (including THs); hence, they are considered ancestors of TRs [126]. TRs can be found in vertebrates and lineages of nonvertebrate deuterostomes [127]. Numerous nonbilaterians can accumulate iodine into specific cells and tissues [125]. However, only chordates have specialized iodine-concentrating organs, the endostyle in cephalochordates and the thyroid gland in vertebrates [126]. Insects, plants, protozoa, and bacteria produce THs (including analogs or derivatives) and deiodinase enzymes [125], even though the incidence of THs might have no hormonal implications outside of chordates. For instance, moon jellyfish Aurelia aurita synthesizes MIT, DIT, and T4 in significant amounts. Nevertheless, the transition from polyp into jellyfish (strobilation) can be induced by temperature and the addition of I − , but is not dependent on THs [128]. Moreover, in land plants, it is suggested that THs, more than hormones, could act as protective agents by altering the metabolisms of herbivores or plagues [129].
Due to iodotyrosines being produced via enzymatic and nonenzymatic reactions, it is no coincidence that THs emerged earlier than other components of the thyroid system. Peroxidases with the potential to iodinate proteins are vastly distributed in prokaryotes and eukaryotes [47], and I 2 may react with organic matter in the environment. Regarding the hormonal role of THs, as organic compounds become more reactive when iodine is incorporated, it is believed that basal chordates developed the endostyle (afterward thyroid) to store iodine in the first place, and animals later took advantage of the reactivity of iodotyrosines produced and incorporated them into their cellular machinery as cell signaling molecules [3,130].

Antioxidant Capacity
Photosynthesis in chloroplasts and the respiratory chain in mitochondria yield multiple reactive oxygen species (ROS), including peroxides (H 2 O 2 is the most common), superoxides (O 2 − ), hydroxyl radicals (OH • ), and O 3 . In appropriate amounts, ROS are involved in host defense, protection from cellular stress, cell signaling, and homeostasis [131,132]. However, some endogen and exogenous stimuli imbalance the amount of ROS, causing irreversible damage to the cells. Under oxidative stress, cellular components and essential molecules (e.g., DNA, RNA, enzymes, and fatty acids) are degraded [131,133]. Thus, cells must produce protective elements such as enzymes and antioxidant molecules (e.g., carotenes and glutathione) to prevent oxidative damage [134][135][136]. Electron donors such as I − can also be considered antioxidants by serving as cofactors for peroxidase enzymes and reducing free radicals [28,137]. In addition, catalytic iodine species can react directly with free radicals by scavenging ROS.
Iodine species having antioxidant capabilities are I − , HIO, and I 2 , with I − being the least reactive and needed for enzymatic assistance to serve as an antioxidant (Reactions (34) and (35)). IO 3 − has no biological activity in the physiological pH range (5)(6)(7)(8). Either way, once ingested, it is converted to I − to be absorbed in the body [24,25]. While most studies focus on the biological activities of I − and I 2 , HIO and its derivatives could also be relevant in studying antioxidant molecules. In water systems, I 2 is converted to HIO via hydrolysis (Reaction (1)) in a fast equilibrium reaction and, in basic pH, becomes the dominant species [24]. Both HIO and I 2 are involved in enzymatic redox reactions as byproducts (Reactions (34) and (35)) [28,90]. Additionally, as oxidizing molecules, they have the potential to reduce peroxides and free radicals by themselves (Reactions (36)- (38)) [138,139], as well as avoid the oxidation of -SH groups in enzymes and proteins (Reaction (39)) [28,61].
To date, photosynthetic marine organisms are the best iodine accumulators, where the primary function of iodine is to control oxidative stress produced by ROS [140]. Laminaria digitata (brown algae) is a hyperaccumulator of iodine that serves for studying iodine metabolism. In this model, the protective role of iodine was demonstrated not only as an antioxidant [141] but also as an element involved in the immune response [142] and maintenance of osmotic balance [143]. L. digitata transports I − in a hydrogen peroxidedependent diffusion manner, quite different from the active transportation in thyroid follicular cells [144]. In the cell wall of brown algae, a vanadium iodoperoxidase (V-IPO) reduces H 2 O 2 to produce HIO (Reaction (34)). Successively HIO can be concentrated in the cytoplasm, be transformed into I 2 , or participate in protecting lipid iodinated compounds ( Figure 5) [141,143]. Many other algae are excellent iodine accumulators, from unicellular organisms such as Cyanobacteria, the first photosynthetic organism known, to Macrocystis pyrifera, a giant kelp. In these marine organisms, it is hypothesized that iodine protects against ROS produced in photosynthesis [1,140]. ular cells [144]. In the cell wall of brown algae, a vanadium iodoperoxidase (V-IPO) reduces H2O2 to produce HIO (Reaction (34)). Successively HIO can be concentrated in the cytoplasm, be transformed into I2, or participate in protecting lipid iodinated compounds ( Figure 5) [141,143]. Many other algae are excellent iodine accumulators, from unicellular organisms such as Cyanobacteria, the first photosynthetic organism known, to Macrocystis pyrifera, a giant kelp. In these marine organisms, it is hypothesized that iodine protects against ROS produced in photosynthesis [1,140]. In land plants, the antioxidant role of iodine is less evident; molecules such as ascorbic acid, polyphenols, carotenoids, and enzymes (e.g., catalase (CAT), guaiacol peroxidase (G-POX), ascorbate peroxidase (APX), and superoxide dismutase (SOD)) are best known for counteracting ROS [146,147]. Nonetheless, in lettuce, tomato, water spinach, and soybean crops, some of these antioxidant markers improve after fertilization with KIO3 and KI [148][149][150].
Non-photosynthetic organisms, including bacteria, fungi, and invertebrates, express different oxidoreductase enzymes with the potential to scavenge H2O2 by using Ias an electron donor [47]. For example, the marine bacterium Pseudomonas iodooxidans has an extracellular peroxidase system that reduces H2O2 by converting Iinto I2. Fungus Caldar- In land plants, the antioxidant role of iodine is less evident; molecules such as ascorbic acid, polyphenols, carotenoids, and enzymes (e.g., catalase (CAT), guaiacol peroxidase (G-POX), ascorbate peroxidase (APX), and superoxide dismutase (SOD)) are best known for counteracting ROS [146,147]. Nonetheless, in lettuce, tomato, water spinach, and soybean crops, some of these antioxidant markers improve after fertilization with KIO 3 and KI [148][149][150].
Non-photosynthetic organisms, including bacteria, fungi, and invertebrates, express different oxidoreductase enzymes with the potential to scavenge H 2 O 2 by using I − as an electron donor [47]. For example, the marine bacterium Pseudomonas iodooxidans has an extracellular peroxidase system that reduces H 2 O 2 by converting I − into I 2 . Fungus Caldariomyces fumago has a chloroperoxidase (CPO) responsible for the oxidation of I − and production of several organic compounds (including iodotyrosines) [32]. Furthermore, as mentioned in Section 5.3.3, moon jellyfish contain THs and other iodinated compounds as a strategy to reduce oxidative stress [128].
Mammals have four classes of peroxidases: TPO, LPO, myeloperoxidases (MPO), and eosinophil peroxidases (EPO), all with the capability of using I − [47]. LPO, MPO, and EPO are involved in the immune response (see Sections 5.5 and 5.6), while TPO is expressed in the thyroid gland, the organ most subjected to oxidative stress. TH synthesis in vertebrates seems paradoxical because organification controls the oxidative stress caused by H 2 O 2 , but TH production demands large amounts of H 2 O 2 [40]. In large part, the activity of TPO reduces the H 2 O 2 produced, but other antioxidant enzymes are needed to protect follicular cells from oxidative damage, such as glutathione reductase, thioredoxin reductase, peroxiredoxins, and CATs [151].

Markers of Oxidative Stress in Mammals
In eukaryotic cells, oxidative stress and other connected biological processes have characteristic biochemical signatures that can be used as molecular markers to evaluate the Future Pharm. 2022, 2 391 oxidative status. In human and animal models, iodine changes oxidative stress markers depending on the chemical species and dosage.
Experimentally, the antioxidant capacity is typically valued as the total antioxidant capacity (TAC), determined by the moles of free radicals scavenged and excluding the activity of antioxidant enzymes [152]. Other common markers include ROS levels, reactive nitrogen species, lipid peroxidation byproducts (e.g., malondialdehyde (MDA)), protein peroxidation byproducts (e.g., advanced lipoxidation end products (ALEs)), reduced/oxidized glutathione (GSH/GSSG), antioxidant enzymes such as SOD and CAT, and other enzymes such as nitric oxide synthase (NOS) [132,153].
As programmed cell death is triggered when cells suffer from oxidative stress damage, apoptosis markers can be considered when studying antioxidants. Apoptosis is characterized by cellular shrinkage, DNA fragmentation, changes in morphology (like reduction in cellular size without losing membrane integrity), activation of caspases (a family of proteases in charge of degrading the cellular structures), etc. [154].
Both, I 2 and I − species have demonstrated antioxidant effects in vitro. In an antioxidant assay (ferric reducing/antioxidant power assay), the TAC of I 2 was 30 to 60 times higher than in KI, Lugol's solution (containing I − , I 2 , and I 3 − ), and IO 3 − . In this assay, I 2 had 10 times more antioxidant power than ascorbic acid, used as a positive control [155]. In a cell-free system, concentrations of 25 µM and 50 µM I 2 (but not 10 µM) significantly reduced the conversion of nitrate (NO 3 − ) to nitrite (NO 2 − ), a reactive nitrogen species [156]. In human sera, the TAC was enhanced by adding 15 µM KI at a comparable level to 50 µM ascorbic acid (vitamin C) [157]. Moreover, KI and sodium selenite (Na 2 SeO 3 ) protected human placenta explants from DNA damage (using 8-hydroxy-2 -deoxyguanosine as a marker) and apoptosis (via Caspase-3 activity) under oxidative stress with menadione (vitamin K3) and antimycin A (an inhibitor of cellular respiration) [158].
Excess I − can be an antioxidant or stressor in animals, depending on the model. In bulls, higher doses of KI in the diet (50% and 100%) improve the TAC, hormonal status (testosterone and T4), and semen quality [159]. A similar observation was registered in boar feed with organic iodine (Prost fodder additive), which increased 43.8% of the blood iodine content and showed better antioxidant, hormonal, and fertility parameters [160]. However, an excess of 100% and 500% in rats (KI 0.7/100 g body weight and 3.5/100 g body weight, respectively) increased oxidative stress markers (lipid peroxidation, SOD levels, and heme oxygenase 1 (HO-1) expression), as well as markers of inflammatory response and apoptosis, while also leading to testicular degeneration changes, reductions in sperm count and motility, and deformities [161,162].

Regulation of Antioxidant Pathways
The response to oxidative stress in eukaryotic cells also involves cell signaling pathways derived in the regulation of the gene expression, such as the Nrf2-ARE pathway. ARE (antioxidant responsive element) is a DNA enhancer sequence involved in the regulation of phase II detoxification enzymes (e.g., NAD(P)H:quinone oxidoreductase-1, glutathione S-transferase, and glutamate-cysteine ligase) [163] and related proteins such as SOD and CAT [164]. The transcriptional factor Nrf2 (nuclear factor erythroid 2-related factor 2) binds to ARE to regulate several target genes. In normal conditions, Keap1 (Kelch-like ECHassociated protein 1) sequesters Nrf2 in the cytoplasm by forming a complex (Nrf2/Keap1). However, under oxidative stress, the complex disassociates, and Nrf2 is translocated to the nucleus, where it binds to ARE (possibly with the transcription factor Maf), upregulating several antioxidant genes [163].
The activity of iodine directly impacts the Nrf2-ARE pathway and, as a result, indirectly influences the cellular response to oxidative stress. In human skin cells, it was demonstrated that I 2 (250 µM) and KI (750 µM) activate the Nrf2-ARE pathway, inducing the expression of CAT and SOD enzymes [165]. It is hypothesized that iodine favors the release of Nrf2 by targeting the cysteine residues of Keap1 and changing its 3D conformation ( Figure 6) [166]. In rats, supplementation with high doses of KI (3.5-5 mg per 100 g body weight) raised the oxidative status in the testis and diminished sperm quality. In affected tissues, a higher expression of oxidative response genes (Nrf2 and HO-1) and related elements (NF-κB and follistatin) was registered, together with apoptotic markers (p53, Bcl-2 proteins, Survivin, cytochrome C, PPAR, Caspase-3, and Caspase-9) [161].
is translocated to the nucleus, where it binds to ARE (possibly with the transcription factor Maf), upregulating several antioxidant genes [163].
The activity of iodine directly impacts the Nrf2-ARE pathway and, as a result, indirectly influences the cellular response to oxidative stress. In human skin cells, it was demonstrated that I2 (250 µM) and KI (750 µM) activate the Nrf2-ARE pathway, inducing the expression of CAT and SOD enzymes [165]. It is hypothesized that iodine favors the release of Nrf2 by targeting the cysteine residues of Keap1 and changing its 3D conformation ( Figure 6) [166]. In rats, supplementation with high doses of KI (3.5-5 mg per 100 g body weight) raised the oxidative status in the testis and diminished sperm quality. In affected tissues, a higher expression of oxidative response genes (Nrf2 and HO-1) and related elements (NF-κB and follistatin) was registered, together with apoptotic markers (p53, Bcl-2 proteins, Survivin, cytochrome C, PPAR, Caspase-3, and Caspase-9) [161].  [166], MDPI, 2016). Abbreviations: I -, iodide; I2, molecular iodine; 6-IL, δlactone; AA, arachidonic acid; ARE, antioxidant response element; CAT, catalase; Cox2, cyclooxygenase type 2; Keap1, Kelch-like ECH-associated protein 1; NOS, nitric oxide synthase; Nrf2, nuclear factor erythroid 2-related factor 2; O2 -, superoxides; OH • , hydroxyl radical; PO, peroxidases; PPARγ, peroxisome proliferator-activated receptor type gamma; PSDD, protein synthesis-dependent diffusion; SOD, superoxide dismutase. The activity of I 2 also results in the synthesis of iodolactones such as α-IHDA and 6-IL from arachidonic acid (AA) [46]. AA is a nonessential fatty acid in the plasma membrane involved in cell signaling pathways. The activation of the AA cascades begins with its release from the plasma membrane via nonenzymatic action of O 2 or via the enzymatic activity of cyclooxygenase (COX), lipoxygenase (LOX), or cytochrome P450 [167]. TPO and LPO can also react with AA and I − to form iodolactones with antioxidant and other significant biological activities, as seen in next two sections [45,168].

Immunomodulation
The immune response can be influenced indirectly by iodine through the regulation of oxidative stress, which could be a consequence of infections and inflammation processes, or via direct participation, such as its role in the LPO system, part of the innate immune response [62]. The enzymatic activity of LPO yields HIO and other ions such as I 3 − and I 2 in the presence of I − [28,64,169]. LPO is secreted in mucosal surfaces or exocrine secretions such as saliva, milk, or tears and is essential for the innate immune defense system [28,47].
Similarly, the leukocyte and eosinophil peroxidase enzymes (MPO and EPO) from white blood cells have the potential to react with I − and produce HIO or I 2 to kill pathogens [170].
Human leukocytes also express components of the thyroid system such as NIS and Pendrin transporters, TG, TPO, and IDs with the potential of modulating TH metabolism [171,172]. The addition of NaI (1 mM) to white blood cells significantly impacted the gene expression and synthesis of immunomodulating agents like immunoglobulin G (IgG), without altering the viability [173]. Upregulated genes include proliferation elements (IL-2, IL-24, and CSF2), inflammatory proteins (IFN-γ, IL-6, IL-1β, and IL-13), and especially chemokines (CCL7, CXCL5, and CXCL6). Likewise, Lugol's solution (I − , I 2 , and I 3 − ) decreases interferongamma (IFN-γ) levels and is involved in both early innate and adaptive immune responses. The treatment with Lugol also increases the release of IL-6, IL-10, and CXCL8 by the immune cells [171].
In the skin, inorganic iodine species revealed an anti-inflammatory potential. As mentioned in the Section 5.4.2, I − and I 2 activate the Nrf2-ARE pathway in human skin cells. Moreover, in ex vivo experiments in human skin explants with induced inflammation (using lipopolysaccharides), only the KI treatment reduced proinflammatory cytokines IL-6 and IL-8. In the same study, skin explants were exposed to 24 h UVB irradiation (300 mJ/cm 2 ), and both I 2 and KI showed a significant protective effect reflected in the higher cell viability and fewer apoptotic events [165].
Iodolipids produced by inorganic iodine species also showed immunomodulatory effects. Ex vivo culturing of follicular cells revealed that 40 times lower doses of 6-IL (0.05 and 0.5 µM) were needed to induce apoptosis in thyroid cells in a similar way to KI (2-20 µM) [174]. The proliferation of thyroid cell lines seems to be regulated by I − and 6-IL by inhibition of the epidermal growth factor (EGF) and inositol 1,4,5-triphosphate (IP3) production (in a dose-dependent manner) but does not impact transforming growth factor beta 1 (TGF beta 1) [175][176][177]. α-IHDA could also be implicit in thyroid function by inhibiting adenyl cyclase, NADPH-oxidase, and TPO in thyroid cells, activating the cyclic adenosine-3 ,5 -monophosphate (cAMP) pathway [178], a master regulator of the adoptive and innate immune cell function [179].
As discussed in the next section, organic and inorganic species' immunomodulatory effects have great pharmacological potential in diseases. For example, in hypothyroid chinchilla with pancreatitis and insulitis (induced by methimazole), supplementation with 0.2 mg/kg I 2 prevents lipoxidation in the serum and pancreas and increases the antiinflammatory response via upregulation of PPARγ receptors. However, a higher dose (2 mg/kg) exacerbates pancreatic damage and induces fibrosis and abnormal amyloid deposits [180].
PPARs are nuclear receptors activated by lipid metabolites (e.g., linolenic acids and prostaglandins) that control the expression of numerous genes involved in inflammation and metabolism. PPARα activates fatty acid oxidation pathways, and PPARγ regulates adipogenesis, lipid metabolism, insulin sensitivity, glucose and lipid homeostasis, and anticarcinogenic pathways [181,182]. Furthermore, 6-IL (produced either with I − or I 2 ) can induce apoptosis and inhibit cell migration of cancer cells in a dependent PPARγ activation mechanism (see Figure 6) [182].

Anticarcinogenic Effect
First observations of iodine as a protective agent were noticed in the Japanese population, whose iodine intake is typically higher and whose lifespan and health status are better than in other countries with lower iodine ingest [65]. The antioxidant capacity of iodine and its impact on several cell signaling pathways could explain these benefits. In this section, the protective role of iodine is addressed in mammal models in the context of cancer and other diseases (excluding the direct intervention of THs).
Higher oxidative status is related to aging and numerous diseases, including thyroid disorders and all types of cancer [98,132]. In these conditions, biological processes such as apoptosis, proliferation, or inflammation are connected, each with characteristic physi-ological phenotypes and specific molecular markers. In normal conditions, apoptosis is promoted when cells suffer irreparable damage. However, cancer cells are characterized by the accumulation of DNA mutations, uncontrolled proliferation, and disruption in caspase activation pathways (e.g., p53 and Bcl-2 pathways), among other hallmarks [154]. These patterns can be modified with iodine supplementation depending on the target (organ, tissue, and specific type of cell), doses, and chemical species used.

Effects on Cells and Tissues
In the literature, three iodine species are best explored as potential anticarcinogenic agents in mammals: I − , I 2 , and 6-IL. Experiments in vitro showed that excessive I − did not affect non-thyroid normal and cancer cell lines (human dermal fibroblasts (PG1), osteosarcoma cells (SaOS), and endometrial carcinoma cells (HeLa)). Contrarily, on immortalized thyroid cells, I − induced apoptosis (seen by morphological changes, DNA fragmentation, and protein crosslinking) and increased oxidative stress (higher ROS and MDA levels). In this study, the programmed cell death process was not dependent on protein synthesis, activation of p53, or proteins from the Bcl-2 family [183]. Proteins from the Bcl-2 family include both antiapoptotic (BCL-2, BCL-XL, BCL-w, MCL-1, and BFL-1/A1) and proapoptotic members (BAX, BAK, BIM, BID, BAD, BMF, and PUMA), and their expression can be linked to promotion or prevention of mitochondrial permeabilization [154].
Molecular iodine, in contrast, exhibits differential activity against breast cells and tissues. Breast cancer cell lines with low and elevated invasive potential (MCF-7 and MDA-MB231, respectively) significantly reduced their viability at 100 to 400 µM I 2 concentrations, but normal epithelial breast cells were not inhibited at the same doses [184]. In a different study, I 2 induced apoptosis in four of five breast cancer cell lines in a timeand dose-dependent manner. In those treatments, mitochondrial membrane potential was dissipated, and expression of Bcl-2 proteins was modified (Bax was upregulated, and Bcl-2 was downregulated) [185]. As caspase activation can be stimulated via two main mechanisms, the extrinsic via activation of death receptors (e.g., TNF or TRAIL) and the intrinsic starting through mitochondrial membrane permeabilization and release of cytochrome C [186], these results suggest that I 2 activates the intrinsic apoptosis pathway mediated by Bcl-2 proteins.
As mentioned, inorganic iodine species can react with AA to generate 6-IL with or without the intervention of peroxidases ( Figure 6) [187]. In breast cancer cells, low doses of 6-IL (5 µM and 10 µM) and higher doses of I 2 (100 µM and 500 µM), but not I − , reduce the proliferation rate [176,187]. In the breast cancer cell line MCF-7, the incorporation of iodine from I 2, but not from I − , to produce 6-IL was demonstrated by autoradiography [187]. Moreover, 6-IL formation (with I 2 ) was related to lower expression of the proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), and urokinase-type plasminogen activator (uPA), while it regulated PPAR type alpha and gamma (PPARα and PPARγ) [188]. Additionally, a higher expression of PPARγ in cervix cancer cells HeLa and SiHa was observed with I 2 (200 µM), together with a lower proliferation and aggregation of the cell lines, as well as a lower expression of stemness genes (CD49f, CK17, OCT-4, NANOG, SOX2, and KLF4), related to self-renewal, generation, and pluripotency [189].

Animal Models and Humans
The effect of iodine species in cancer progression has also been studied in animals after tumor induction with drugs or implantation of exogenous abnormal cells (xenografts). In Sprague-Dawley rats with breast-induced tumors (with N-methyl-N-nitrosourea), the absorption of radioisotopic 125 I − and 125 I 2 was comparable. However, the incidence in tumors was different when the drinking water was supplemented with I − , I 2 , and T4. In the long term (16 weeks), continuous supplementation with I 2 exposed lower MDA levels and lower incidence of cancer (30%) than KI (93.7%), T4 (81.8%), or controls (72.7%). Serum T3 or NIS, LPO, and the p53 gene expression were not affected in I 2 treatments [190].
On immunodeficient animals (athymic nude mice) with implanted human breast cancer cells, the incidence and size of tumors were significantly lower under I 2 supplementation (25-50% and 0.4-0.5 cm 3 , respectively) compared with nonsupplemented controls (91.6% and 1.4-2.4 cm 3 , respectively). In this study, animals who drank water with I 2 maintained normal body weight gain, expressed higher levels of PPARγ, and had decreased expression in the proliferating cell nuclear antigen (PCNA) [184], a marker of proliferation in breast cancer diagnosis [191].
Furthermore, I 2 has demonstrated adjuvant properties when supplemented with chemotherapy. Mice with neuroblastoma-induced cancer (using the cell line SK-N-BE(2)) had a preventive effect on bladder damage and a similar reduction in tumor size with I 2 (8 mg/kg/day) and chemotherapy (cyclophosphamide 20 mg/kg/day)). At the molecular level, all the treatments with I 2 downregulated the expression of proto-oncogenes (MYCN and TrkB) and upregulated genes associated with neuronal differentiation (p53, PPARγ, TrkA, and Bax) [192]. Furthermore, doxorubicin chemotherapy in combination with I 2 (3-6 mg/day) had the best anticarcinogenic effect and heart protection in female rats with induced mammary cancer (using methyl nitrosourea). Treated rats showed a significantly developed tumor size and lower proliferation rate, together with reduced MDA levels in the heart. Noticeably, I 2 suppresses tumor chemoresistance in the long-term by downregulating Bcl2 and survivin genes and upregulating Bax and PPARγ expression [155].
Induced benign prostate tumors in rats had a comparative antioxidant and antiproliferative improvement after supplementation with I 2 (2.5 mg/day/rat) and celecoxib (1.25 mg/day/rat), an anti-inflammatory drug [156]. I 2 supplementation and celecoxib prevented lipoxidation and production of reactive nitrogen species (NO 2 − ). Likewise, I 2 repressed the expression of the PCNA gene and inhibited pro-oxidant NOS and proinflammatory Cox2 enzymes. Cox2 generates prostaglandin H2 from AA, especially during inflammatory processes [193]. Overexpression of Cox2 is related to carcinogenesis in the prostate and other organs [194]; hence, its inhibitors potentially improve cancer progression. The anti-inflammatory and antiproliferative mechanisms of iodine related to COX expression could be complex to address, but they can be initiated by simply reducing available AA and preventing prostaglandin production by producing 6-IL.
In humans, there was one randomized pilot study in women diagnosed with early and advanced breast cancer (stages II and III, respectively) treated with I 2 (5 mg/day) and I 2 combined with a chemotherapeutic cocktail (5-fluorouracil/epirubicin/cyclophosphamide or taxotere/epirubicin). In all treatments containing I 2 , the chemoresistance and side-effects improved in the patients. Most important, the 5 year survival rate was significantly higher in chemotherapy combined with I 2 than alone (82% and 46%, respectively). Transcriptional responses under I 2 and I 2 chemotherapy included upregulation in pathways related to inflammation, immune response, and survival, while antiapoptotic and chemoresistance pathways were downregulated [195]. Further transcriptomic analysis of the treated tumors revealed a higher antitumor response in I 2 supplementation alone or with chemotherapy by activating the Th1 differentiation pathway (involved in eliminating pathogens and cancerous cells). The immune cells profile in the I 2 treatments showed cytotoxic activity of natural killer and CD8 + cells and higher infiltration of nonactivated macrophages and B lymphocytes [196].

Additional Considerations
A key feature in the biological activity of iodine is the expression of transporters on the surface of target cells. The main transporters of I − are NIS and Pendrin, but there are others such as ANO1, CFTR, and SMVT [94,95]. To date, it is known that I 2 is not transported by NIS, Pendrin, or TPO activity but in a protein synthesis-dependent diffusion manner [187,197]. So far, no recent evidence supports the passive diffusion of I 2 or any iodine species.
In normal conditions, the thyroid, kidney, salivary glands, lactating mammary glands, and placenta express higher levels of I − transporters [97,166]. Contrarily, in cancer, the profile change, and the breast, colon, pancreas, liver, ovary, and skin tumors overexpress I − transporters. In the prostate, normal cells better express Pendrin, but NIS and CFTR dominate in the tumoral tissues [97]. The differential expression of iodine transporters could have a practical application in diagnostics. In ovary and nasopharynx cancers, the increment in CFTR expression is correlated to cancer progression and is proposed as a prognostic marker [198,199]. Moreover, higher expression of NIS and Pendrin is seen in women with a history of recurrent abortions (≥2) and could be a marker to predict adverse pregnancy outcomes in women with a subclinical IDD [200].
An important implication of aberrant expression of ion transporters is changing tissular pH balance. Normal cells have an intracellular pH value of~7.2 and~7.4 extracellularly. In cancer, these parameters are altered, resulting in a pH gradient from a more acidic pH in the outer space (~6.7-7.1) to an alkaline environment inside the cells (>7.4) [201]. Altered pH is probably the less described hallmark in cancer research and one of the most influential variables in the chemical speciation of iodine and, consequently, a relevant feature when studying its anticarcinogenic activity.
Recent studies have shown promising results with high doses of I 2 (such as 5 mg/day) in the treatment of cancer (especially in the breast) [195]; however, this biological activity must imply the presence of I − and possibly HIO. This is because, firstly, I 2 has poor solubility in water without additional iodide (0.293 g/L) [21] and, secondly, its chemical speciation in water systems renders I − and, depending on pH, also HIO (Reaction (1)) [24]. As HIO is formed from I 2 in a swift reaction at pH ≥7 [24], the contribution of HIO and probably some of its derivatives (such as OI − ) inside the tumoral cells could be implicit and must be taken into account [24].
Furthermore, similar anticarcinogenic effects have been observed with 6-IL and I 2 ; thus, it is necessary to consider the formation of organic iodine species (iodoproteins and iodolipids) produced by its catalytic potential. For example, in breast tumors, the composition of the cell membrane is different than in normal cells. In particular, there is a higher content of AA and PPARα [202]. Thus, a feasible explanation of the antitumoral action of I 2 might also be given by its conversion to 6-IL. Similarly, the protective effects of I − in the thyroid and other tissues expressing peroxidases could be partly mediated by its incorporation into lipids.

Iodine in the Industry
Molecular iodine is one the most exploited chemicals in the industry, mainly as a catalyst for organic synthesis (including many heterocyclic compounds used in pharmacy), because of its low cost, efficiency, and high selectivity [203]. I − is considered a mild reducing agent, and it is a much less reactive species than I 2 . IO 3 − is less reactive and has low toxicology; together with I − , it can also be used as a chemical reagent or serve as an ingredient for various products (e.g., supplements and iodophors) [8,204]. Hydrogen iodide (HI), iodine pentoxide (I 2 O 5 ), iodic acid (HIO 3 ), and sodium periodate (NaIO 4 ) are other frequent chemicals used in the pharmaceutical industry for organic synthesis [205,206]. In this review, the chemical synthesis is not addressed, but it is recommended to consult Historically, Lugol's solution has been one of the most used iodine-based products in medicine and the industry. It was first used in the early 20th century by J.G.A. Lugol as a treatment for tuberculosis and is still considered an essential component of basic healthcare systems [207,208]. Lugol's solution is used as an antiseptic, disinfectant, supplement, and laboratory reagent, among the most important applications [2,29]. Currently, many iodinebased products are being developed, and the global demand for iodine has increased in the last decades. In 2017, the iodine market value was 833 million USD, and it is forecasted to be~1.14 billion USD by 2024 [209].
Chile and Japan lead the global production of iodine (having also the biggest reserves worldwide), followed by Turkmenistan, Azerbaijan, Indonesia, and Russia (the US has been excluded from statistics in recent years) [210,211]. In the case of radioisotopes (used in nuclear medicine), it must be produced in nuclear reactors or cyclotrons from tellurium or xenon gases [212,213]. The major industrial applications of iodine include production of X-ray contrast media (XCM) (22%), pharmaceuticals (13%), polarization films (12%), animal feed (8%), iodophors (7%), fluorochemicals (7%), biocides (4%), nylon (4%), and human nutrition supplements (3%) [214]. The section below describes the relevant iodine applications of iodine in biomedicine and some of the representative iodine-based products.

Disinfection, Asepsis, and Wound Care
The first iodine-based tinctures, such as Lugol's solution, became popular as disinfectants because of their nonselective antimicrobial action against a broad range of microorganisms. However, the use of these kinds of products in drinking water disinfection is not recommended because of the risk of undesirable byproducts. More than 600 peptides are found in water [215], and reactions of chloramine (NH 2 Cl), H 2 OI + , and/or HIO with aromatic peptides (e.g., dipeptide Tyr-Gly) form highly toxic iodinated compounds of concern for people and the environment (Reaction (40)) [216]. Reactions of HIO, I 2 , OI − species, and chloramines also affect pyridine nucleotides and glutathione (GSH), a potent and essential cellular antioxidant molecule (Reaction (41)). HIO (resulting from I 2 and OH − ) is a more selective downgrader of reduced dihydro-nicotinamide mononucleotide (NMNH) and nicotinamide adenine dinucleotide phosphate (NADH) than HOCl, and it can directly oxidize GSH or decrease its production by reducing the availability of NADPH (needed in GSH cycling) [217].
The use of iodine-based products in surgical procedures is extended. Nonetheless, classic Lugol's solution has some drawbacks, such as staining of the skin and materials, local pain, and irritation. In addition, a proportion of I 2 , the main component responsible for the antimicrobial activity, is lost by volatilization. These problems were solved in the early 1950s by the development of iodophors. The advantages of iodophoric preparations are reduced staining, increased I 2 solubility, and slow release to prolong the antimicrobial action [25,218]. Iodophors are made with solubilizing agents such as natural or synthetic polymers or surfactants, and they can be anionic, cationic, or nonionic [24,219]. Natural polymers are mostly starch and cellulose derivatives (e.g., amylose, amylopectin, Cadexomer, carboxymethyl cellulose, and methyl hydroxypropyl cellulose). Chitosan and lecithin are used in more complex iodophoric formulations (e.g., carboxymethyl chitosan and hydroxylated lecithin). Regarding synthetic polymers, PVP/I is the most used, followed by polyvinyl alcohol with iodine (PVAI). Other excellent polymeric carriers include poly(4-vinyl pyridine), poly(3-vinyl-10-methylphenothiazine), poly(2-ethyl-2-oxazoline), and poly(tetramethylene ether) glycol [219,220].
PVP/I was developed in 1956 and is suitable for wound healing because it is well tolerated on inflamed tissue, acting in a pH range of 2.5-7.0. It is also extensively applied as a disinfectant in the industry. For instance, PVP/I is routinely used as a milk and teat disinfectant and is, in fact, an important contributor to the iodine content in dairy products [221]. In the past, PVP/I was associated with bacteremia [222][223][224], but it was later demonstrated that contamination came from stock solutions. When working solutions were prepared according to the manufacturer's instructions, in all cases, they effectively inactivated all bacteria isolated form nosocomial infections [225,226].
Iodophors have also been exploited in novel wound healing and anti-inflammatory products because of their biocompatibility. PVAI and PVP/I-based hydrogels are good examples; they are constituted by flexible matrices that maintain asepsis and moisture. Moreover, they also absorb exudates, allow wound oxygenation, and have low toxicity and skin irritation [54,237]. Together with other components, hydrogels can serve as scaffolds for tissue regeneration, regulate medication released, or display unique properties to respond to stimuli, such as color change by temperature (thermochromism) or pH/thermal sensitivity [238,239].

Supplementation
When iodine ingestion is insufficient, there is a need to consume supplements to maintain normal thyroid function (euthyroidism). Iodine intake by groups of age is recommended as follows: 90 µg/day for infants and children 0-59 months, 120 µg/day for school children (6 to 12 years), 150 µg/day for adults, and 200 µg/day for pregnant and breastfeeding women [240]. Upper limits are much more difficult to establish because they are influenced by previous long-term iodine status [84] and even the kind of supplementation [166]. Some studies reported normal thyroid function at high doses of iodine from the diet (mainly from seaweed, 1.2 mg/day) [241], supplements (1-3 mg/day) [242], Lugol's solution, and PVP-based water disinfectants (4-8 mg/L water) [243]. However, the recommended safe iodine intake for euthyroid people by the WHO is up to 1 mg/day, except for pregnant and lactating women that have no extra benefit receiving >500 µg/day and children of <2 years that should not exceed >180 µg/day [244].
The most common and suitable indicator to evaluate the iodine status in a population is urinary iodine, given that iodine is mostly excreted in urine (~90%). In this sense, the best method is urinary iodine excretion (UIE), which is determined from the whole urine collected for 24 h. Urinary iodine concentration (UIC) from one urine sample (preferably collected in the morning) is enough and more practical to define the general status according to WHO [2013].For most people (including lactating women), >100 µg/L UIC is considered adequate, but <100 µg/L UIC reflects insufficient intake (<20 µg/L UIC suggest severe iodine deficiency), except for pregnant women whose optimal UIC is considered 150-249 µg/L, with <100 µg/L considered insufficient [245]. Nonetheless, urinary concentrations might vary in short periods (days-hours). Thus, to determine iodine status and thyroid function of individuals, it is recommended to screen the levels of TSH and free T4 as a minimum [66,246]. Other parameters are evaluated to monitor thyroid function, such as goiter rate (%), levels of T3, rT3, TG, antibodies for TG (TgAb) or TPO (TPOAb), and thyroid-stimulating immunoglobulin (TSI) [242].
Optimal consumption of iodine can be obtained with an appropriate diet, consuming seafood (e.g., seaweed, fish, crabs, and oyster), dairy products (e.g., milk, yogurt, and cheese), tubers (e.g., turnip and saffron), beef kidney, green beans, eggs, yeast, some condiments (e.g., chili powder and mint), and iodized salt [39,247]. However, some geographical areas might have iodine-deficient water and soils; consequently, the iodine content in food is lower. For example, iodine content in plants can be up to 1 mg/kg or down to 10 µg/kg (dry weight), depending on the soil [66]. Geographic iodine-deficient areas include mountainous and inland regions of South America (e.g., the Andes and inland Brazil), Midwestern United States, Europe (e.g., Alps, Pyrenees, England, Wales, Greece, and the Netherlands), Asia (China, India, Bangladesh, Himalayan hillsides, and Indonesia), Africa (e.g., Atlas Mountains, Nigeria, Cameroon, the Central African Republic Democratic Republic of Congo, Uganda, and Ethiopia), Southern Australia, and Highlands of New Guinea [65].
The additional intake of iodine from supplements is recommended to prevent IDD in iodine-deficient areas (especially among women of reproductive age). Currently, the best strategy to face IDD in the world has been the universal iodization of table salt (with KI, NaI, or KIO 3 ), implemented in 1994 by the International Council for the Control of Iodine Deficiency Disorders (ICCIDD). The advantages of this product lie in the global consumption of salt, cheap cost production, long shelf-life, accessibility for remote communities, and imperceptible taste modification [248]. IDD persists even in developed countries due to the unequal consumption and distribution of iodized salt among low-and high-income households [249,250]. Moreover, it has been demonstrated that iodine can be partially or entirely lost during cooking depending on cookware materials and ingredients such as ascorbic acid, glucose, and some condiments, as well as because of impurities and storage conditions, mainly the kind of package and humidity [251,252].
In the market, there is a wide variety of supplements that can also be taken with one or more of the following components in their formulations: KI, NaI, KIO 3 , I 2 , iodized oil, natural products based on seaweed (from species Laminaria spp., Fucus vesiculosus, Alaria esculenta, Ascophyllum nodosum, Ecklonia maxima, Eisenia bicyclis, Hizikia fusiforme, Palmaria palmata, Porphyra tenera, Postelsia palmaeformis, and Sargassum sp.) or plants (e.g., Commiphora mukul and Iris versicolor) [39,253,254]. Among all supplements, natural formulations have the most variability or declare the highest iodine contents and should be avoided for pregnant women or those planning a pregnancy [254,255]. Natural or synthetic thyroid hormones are used not as supplements but under prescription to treat hypothyroidism. Currently, levothyroxine (or L-thyroxine) is the most recommended by endocrine societies (and produced worldwide) for thyroid hormone replacement therapy [256].
Probiotic consumption might improve iodine status, given that the gut microbiome participates in nutrient absorption and cycling of THs. Autoimmune thyroid dysfunctions and thyroid carcinoma are linked to dysbiosis (imbalance in the microbiota). Hypothyroidism and hyperthyroidism are correlated with the reduction in intestinal bacteria from Lactobacillaceae and Bifidobacteriaceae families [257].

Contrast Agents
X-rays allow us to see structures of high density because the X-ray absorption coefficient (µ) is directly proportional to the atomic mass of the compound. Bones and some organs in the body can be directly distinguished by X-ray, but other less dense structures need contrast agents to visualize them [261]. X-ray contrast media (XCM) are substances that allow visualization of those biological structures, and, depending on the difference in µ, they can be classified as positive or negative. Negative XCM are gases of lower absorption coefficient (e.g., O 2 and CO 2 ), and positive XCM contain elements of high atomic number, such as iodine or barium, that stand out in X-ray imaging [261,262].
Positive XCM can be classified as targeted (or specific) and extracellular (or unspecific), depending on whether the contrasting reagent is metabolized in the body [15,261]. Extracellular XCM are water-soluble agents mainly used to visualize the circulatory system. The pharmacokinetics is very similar among extracellular XCM, taking 3-10 min for distribution and typically 1-2 h in elimination (via glomerular filtration). Under renal impairment, the excretion takes up to 10 h with a higher risk of nephrotoxicity [15,261].
On the contrary, targeted XCM (also biliary contrast agents) have good lipophilicity and different distribution and elimination times. The disadvantages of XCM with low hydrophilicity are less tolerability, hepatic or renal toxicity, and higher allergic reactions [261,263]. For those reasons, research on targeted XCM is now developing into noniodine compounds, although promising advances have been accomplished with liposomes and polymers as carriers [263].
The simplest contrast agent is NaI, the first iodinated XCM in the market (in 1918), with an excellent µ value but not as good tolerability at high doses. Currently, most XCM are based on molecules of one or more triiodobenzene rings. Triiodobenzene derivatives are the extracellular XCM of choice because of their stability, tolerability, low toxicity, and wide range of compounds that can be produced. The synthesis is achieved by substituting the hydrogens at positions 1, 3, and 5 of the benzene ring with I atoms. Some good examples are iohexol, iopamidol, ioversol, ioxilan, iopromide, ioxaglic acid, iotrolan, iodixanol, and diatrizoate meglumine [264]. Examples of approved targeted agents are iodipamide meglumine to visualize the bile duct and the gallbladder and lipiodol for imaging hepatocellular carcinoma [265][266][267].

Applications in Nuclear Medicine
All kinds of radiation can be dangerous for life, but the extent of damage depends on the decay mode and half-life of the radioactive atom. Alpha (α) and beta (β) radiation are weaker but significantly dangerous when particles are inhaled, swallowed, or absorbed in the body. Gamma (γ) and X-rays are much more powerful and penetrating, causing immediate severe cellular damage from external sources [268]. In a controlled fashion, nuclear medicine takes advantage of radioisotopes that can be detected in small amounts and represent a lesser danger for life, to diagnose and treat several diseases.
Diagnostics in nuclear medicine differ from radiology in the study of the functionality of tissues and organs, not only examining anatomic structures. For this purpose, radiopharmaceuticals are injected (most of the time), inhaled, or orally administered to follow their circulation, accumulation, or metabolization in the body [261,269]. For imaging, developed technologies are scintigraphy, single-photon emission computed tomography (SPECT), positron emission tomography (PET), or combinations with computed tomography (CT) such as SPECT/CT and PET/CT [269][270][271].
Radiotherapy also uses radioisotopes to target specific abnormal cells or tissues and destroy them via internal radiation (e.g., hyperthyroidism caused by Graves' disease toxic nodular goiter) [272]. The most used radioisotope for both diagnostics and therapy is technetium ( 99 Tc), followed by iodine, because of their high intensity and short halflife [269,272]. The main iodine radioisotopes of iodine exploited in medicine and for research are 123 I, 124 I, 125 I, and 131 I [273]; their physicochemical properties are described in Table 1.
The simplest agent containing an iodine radioisotope is NaI, which can be orally ingested to evaluate thyroid and parathyroid functioning (using 123 I or 124 I) or to treat thyroid disorders and thyroid cancer (with 131 I) [264,272]. Additionally, radioactive iodides can serve to label molecules by nucleophilic substitution (usually benzene rings derivatives), switching stable iodine ( 127 I) with the radioisotope [269]. Some pharmaceuticals having aromatic rings are meta-iodobenzylguanidine (MIBG), a norepinephrine analog (with 123 I, 124 I, or 131 I) for diagnosis of pheochromocytoma, neuroblastoma, and heart diseases; ioflupane 123 I (a cocaine analog) for detection of Parkinson's disease and dementia; iothalamate 125 I to evaluate glomerular filtration; ioetamine 123 I to detect ischemic brain diseases, and iodohippurate 123 I to diagnose kidney dysfunction [264,275]. Radiotherapy and imaging can combine (theranostic) by using a single agent for visualization and treatment of abnormal cells. For example, by taking advantage of the high accumulation of I − in the thyroid, Na 131 I is administrated to image and treat thyroid cancer and hyperthyroidism. Furthermore, 123 I and or 131 I combined with metaiodobenzylguanidine (MIBG), a neurotransmitter analog, is used to treat neuroendocrine tumors [276,277]. Furthermore, theranostics might involve gene and cell-based therapy to deliver pharmaceuticals to specific cells. For example, the increment in NIS expression (by gene transfer) in non-thyroid tissues, such as in the cervix and prostate tumors, facilitates the entry of radioisotopes of iodine for inducing abnormal cell death [278,279]. Other therapeutic approaches include labeled antibodies such as 131 I tositumomab, approved for non-Hodgkin's lymphoma treatment, labeled nanoparticles coated with metals (e.g., gold or copper sulfide) used in SPECT/CT, or even devices such as 125 I seeds (within titanium capsules) used in inoperable lung tumors and nonpalpable mammary lesions, among others [272,[280][281][282]. 124 I has not been used as a routine agent in diagnostics mainly because of its complex decay mode and longer half-life. However, it can be imaged by PET with more sensibility and 3D acquisition in a quantitative manner. Some interesting radiopharmaceuticals developed with it are A14-iodoinsulin 124 I, FIAU iodouracil 124 I, and AntiCEA minibodies 124 I, which exhibited promising results for the treatment and diagnosis of tumors [271].

Prophylaxis
Radioactive contamination after a nuclear explosion or an accident can bring severe consequences for people and the environment in the short and long term, as occurred with the atomic bombings of Hiroshima and Nagasaki in 1945 or the Chernobyl (former Soviet Union) and Fukushima (Japan) nuclear accidents in 1986 and 2011, respectively. Beyond the immediate damage of radiation, iodine radioisotopes are of particular concern because of their accumulation in the body and potential incorporation into the food chain following regular iodine cycling [283][284][285]. High radiation levels can induce hypothyroidism or acute thyroiditis. Lower exposure to radiation increases the risk of thyroid cancer and benign thyroid nodules in the long term, especially in infants, children, and adolescents [69].
To prevent the absorption of radioactive isotopes (prophylaxis), high doses of stable iodine ( 127 I) can be promptly distributed in the population [242]. Prophylaxis is not used to restore iodine status; instead, it takes advantage of the Wolff-Chaikoff effect, a transient inhibition of TH production induced by excess iodine. In most cases, the thyroid function is restored in 1-2 weeks but vulnerable groups (e.g., fetuses, neonates, and patients with autoimmune thyroiditis and Graves' disease or treated with antithyroid drugs) may not "escape" from this effect [286].
Prophylaxis is recommended when exposure to vulnerable groups (neonates, infants, and children) is ≥10 mGy (1 Gy is equal to 1 J of radiation absorbed per kg) [287]. Older adults (>40 years) have a minimal thyroid cancer risk and more side-effects with excessive iodine supplementation; therefore, prophylaxis is recommended at ≥100 mGy. According to WHO, a single dose of I 2 , KI, or KIO 3 is sufficient for all groups of age, as indicated in Table 2. Repetition doses are only prescribed for pregnant women from iodine-deficient areas and vulnerable people with prolonged exposure (for example, infants inhaling radioactive material) [287].

Conclusions
The chemical speciation of iodine in aqueous systems is complex and influenced by pH, redox potential, dilution, and the occurrence of chemical compounds. In this universe, four inorganic species have leading roles in biological systems: I − , I 2 , IO 3 − , and HIO. Iodate represents a major source of iodine and is well absorbed in several organisms. However, it only achieves some reactivity at extremely low pH; hence, it needs to be transformed into a different species to exhibit a more active role. HIO and its derivatives are reactive iodine species with excellent antimicrobial and antioxidant properties; however, their amount and stability are limited or negligible under pH 7. In contrast, I 2 is also a reactive species that can be predominant in conditions compatible with life. I 2 has poor solubility in water; hence, it needs to be combined with I − to increase its availability. Lastly, I − has good water solubility, and it is probably the most versatile of all. I − is not as reactive as I 2 or HIO but can be used as an electron donor in several enzymatic reactions, yielding numerous inorganic and organic species. I − and I 2 are constantly cycled in the environment and can be linked to virtually all known biological processes involving iodine. Thus, both can be considered the main inorganic species in the biological context.
Iodine plays a fundamental role as an essential nutrient needed in all stages of life. Its deficiency has severe consequences for human health, from congenital anomalies in the prenatal and neonatal stages to severe metabolic disorders. On the contrary, iodine in higher doses can significantly improve health status by increasing the antioxidant capacity and innate immune response. Certain iodine species also show anticarcinogenic and immunomodulatory activities in some types of cancer. Moreover, after nuclear disasters, one single very high dose has the potential to protect the population from iodine radioisotopes released into the environment.
In general terms, the biological activities of iodine can be divided into (1) iodination of organic compounds (e.g., TH synthesis), (2) degradation of molecules (including neutralization of ROS and antimicrobial activities), and (3) intervention in cell signaling (from hormonal roles to cell signaling regulation). Each has a direct application in medicine, biology, biotechnology, and many other fields.
Even though hormone synthesis has the protagonist role in the performance of iodine, there is sufficient evidence to support that inorganic species are as important not only in supplementation but as active elements in the evolution of life as we know it. Receptors, TSH Thyroid-Stimulating Hormone, Tyr Tyrosine, UIC Urinary Iodine Concentration, VEGF Vascular Endothelial Growth Factor, V-IPO Vanadium Iodoperoxidase, XCM X-ray Contrast Media, 6-IL δ-lactone, α-IHDA α-Iodohexadecanal, β − Beta particle emitter, β + positron emitter, ε Electron capture.