The protective role of nutritional antioxidants against oxidative stress in thyroid disorders

An imbalance between pro-oxidative and antioxidative cellular mechanisms is oxidative stress (OxS) which may be systemic or organ-specific. Although OxS is a consequence of normal body and organ physiology, severely impaired oxidative homeostasis results in DNA hydroxylation, protein denaturation, lipid peroxidation, and apoptosis, ultimately compromising cells’ function and viability. The thyroid gland is an organ that exhibits both oxidative and antioxidative processes. In terms of OxS severity, the thyroid gland’s response could be physiological (i.e. hormone production and secretion) or pathological (i.e. development of diseases, such as goitre, thyroid cancer, or thyroiditis). Protective nutritional antioxidants may benefit defensive antioxidative systems in resolving pro-oxidative dominance and redox imbalance, preventing or delaying chronic thyroid diseases. This review provides information on nutritional antioxidants and their protective roles against impaired redox homeostasis in various thyroid pathologies. We also review novel findings related to the connection between the thyroid gland and gut microbiome and analyze the effects of probiotics with antioxidant properties on thyroid diseases.


Introduction
Cellular redox homeostasis depends on a dynamic equilibrium between prooxidant production and its elimination. Reactive oxygen species (ROS), together with reactive nitrogen species (RNS), represent the most important prooxidants whose excessive accumulation leads to oxidative stress (OxS) and molecular damage (1,2). ROS are molecules with an oxygen atom, and unpaired electrons are primarily generated as by-products of ATP synthesis in mitochondrial respiratory chains (1) or during inflammation (3,4). The concentration of ROS determines their physiological role (5). When present at low concentrations, ROS are involved in signaling processes essential for normal cellular functions (6,7), whereas high ROS concentration leads to DNA, lipid, and protein damage and apoptosis (8).
ROS are crucial in thyroid function because they are essential in the initial stages of thyroid hormone synthesis during iodide oxidation (9). Also, the process whereby thyroid peroxidase (TPO) catalyzes thyroxine (T4) and triiodothyronine (T3) during its synthesis in thyroid follicles involves ROS (10). In addition, thyroid hormones affect the mitochondrial activity and modulate ROS production (10). The dependence of normal thyroid function on ROS implies that the thyroid is continuously exposed to ROS and, thus, particularly sensitive to oxidative damage (10). Therefore, to protect the integrity of the thyroid, it is mandatory that the thyroid antioxidant defence system effectively regulates and balances ROS production and elimination (11,12).
Aerobic organisms have evolved multiple antioxidant and repair systems for protection against OxS. Enzymes that decompose ROS, such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) provide the primary antioxidant defence (5,7,8,13). In contrast, ROS-induced damage repair systems eliminate damaged cells through autophagy and apoptosis processes (14,15). However, the capacity of intrinsic antioxidant systems is not always sufficient to prevent damage caused by excessive accumulation of ROS. Thus, non-enzymatic mechanisms based on the action of molecules with antioxidant properties such as glutathione (GSH), thioredoxin, coenzyme Q10, and exogenous antioxidants, including various polyphenolic compounds, ascorbic acid, tocopherol retinol, and b-carotene, that may also support antioxidant systems are essential. The use of nutritional antioxidants as supplementary substances that delay and/or prevent the oxidation of cellular components has shown the potential to protect human organs, including the thyroid gland, against oxidative damage by reinforcing the body's antioxidant defence and increasing total antioxidant capacity (5,16,17).
Recently, a search for natural nutritional antioxidants from biological resources has gained substantial attention. Of particular interest are probiotics which represent live nonpathogenic microorganisms that can restore microbial balance in the gastrointestinal tract upon appropriate administration (18). Evidence demonstrates that probiotic bacteria exert significant antioxidant effects in vitro and in vivo (19)(20)(21)(22), and the connection between the thyroid gland and gut microbiome is well-established. Furthermore, it has been documented that dysbiosis, an imbalance in gut microbiota, is associated with impaired thyroid function and pathogenesis of thyroid disorders such as Hashimoto's and Graves' disease (23). In this review, we discuss the protective role of exogenous nutritional antioxidants in the context of various thyroid disorders. We also review novel findings related to the connection between the thyroid gland and gut microbiome and analyze the effects of probiotics with antioxidant properties on thyroid diseases.

Search strategy
We searched MEDLINE and PubMed for all English and non-English articles with English abstracts published between 1977 and 2022. The leading search terms were: oxidative stress, reactive oxygen species, thyroid disease, nutritional antioxidants, thyroidgut axis, gut microbiome, and antioxidant probiotics. The search retrieved original peer-reviewed research articles, which were further analyzed, focusing on the role of nutritional antioxidants in thyroid diseases. We specifically focused on including the most recent findings published in the past five years.

Oxidative stress
Oxidative stress is a disbalance caused by excessive production of prooxidant substances such as ROS and RNS and/or the antioxidant systems working inefficiently (14,24,25). ROS include superoxide anion, hydroxyl radical, and hydrogen peroxide, which are produced in vivo primarily by the mitochondrial respiratory chain during aerobic metabolism (26). RNS family includes peroxynitrite, generated via a reaction between nitric oxide (NO) and superoxide, and nitrosoperoxycarbonate, generated via a reaction between peroxynitrite and carbon dioxide. Under physiological conditions, ROS plays a vital role in maintaining cellular homeostasis by regulating the endogenous antioxidant pool (27-30) and participating in host defence and hormone synthesis (31, 32). In thyrocytes, ROS production is essential for their functional role (33) since TPO-mediated hormone synthesis depends on the action of dual oxidases (DUOX), enzymes responsible for H 2 O 2 production (34). However, when ROS and RNS are present in excessive amounts and/or in the form of highly reactive free radicals such as superoxide anion and hydroxyl radical, they oxidize susceptible biomolecules such as membrane lipids, cellular proteins, and nucleic acids, leading to disruption of normal cellular functions (35). Lipid peroxidation is a process in which oxidants such as free radicals or non-radical species attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs) (36). The main lipid peroxidation products are hydroperoxides, such as propanal, hexanal, 4-hydroxynonenal, and malondialdehyde (MDA) (37). Phospholipids, cholesterol, and glycolipids are also targets of potentially lethal peroxidative modifications (38). ROS can also cause damage to DNA by oxidizing nucleoside bases (39). For example, guanine oxidation produces 8-oxo guanine (8-oxoG), which may lead to G-T or G-A transversions if unrepaired. The guanine and deoxyguanosine oxidation products 8-oxoG and its nucleotide 8-oxo-2′deoxyguanosine (8-oxodG) are ROS-mediated DNA lesions considered the most significant biomarkers for oxidative DNA damage (40). Oxidized bases are usually recognized and repaired by the base excision pathway (BER). Still, when they co-occur on opposing strands, BER can lead to the generation of doublestranded DNA breaks (41). ROS accumulation also induces mitochondrial DNA lesions, strand breaks, and DNA degradation (42). In addition, increased ROS levels are responsible for protein oxidation that can rapidly contribute to the augmentation of OxS by directly affecting cell structure, cell signaling, and essential enzymatic metabolic processes. Several modes of ROS-mediated protein oxidation are reported, including metal-catalyzed oxidation, oxidation-induced cleavage, amino acid oxidation, and the conjugation of lipid peroxidation products (43).
Excessive ROS accumulation is an important factor in the pathogenesis of different diseases. For instance, an elevated ROS production by the respiratory chain is observed in obesity as a response to metabolic overload caused by excess macronutrients and increased substrate availability (44). Mitochondrial dysfunction and endothelial reticulum stress contribute to metabolic perturbances in the adipose tissue of obese patients (45). Consequent ROS accumulation leads to cell damage and pathogenesis of inflammatory and cardiovascular diseases (46). Furthermore, mitochondrial ROS acts as signaling molecules mediating pro-inflammatory cytokines' production, further reinforcing the connection between OxS and inflammation (47).
Several enzymatic and non-enzymatic defence mechanisms that guard cells against free radical damage have been identified in different cellular localizations, including mitochondria, plasma membrane, endoplasmic reticulum, peroxisomes, and cytosol. For example, enzymes SOD, Cat, and GPx, and transition-metal binding proteins, such as transferrin, ferritin, and ceruloplasmin, inactivate free radicals (48). Three forms of SOD are known in mammals: cytoplasmic SOD (SOD1), mitochondrial SOD (SOD2), and extracellular SOD (SOD3) (49). SOD belongs to a group of metalloenzymes that catalyzes the dismutation of superoxide anion to hydrogen peroxide and molecular oxygen, while Cat decomposes hydrogen peroxide to water and molecular oxygen (50). In high H 2 O 2 levels, GPx also participates in detoxification by converting lipid peroxides to the corresponding alcohols. Hydrosoluble molecules with free radical scavenging properties such as ascorbic acid, albumin, bilirubin, urates and thiols, liposoluble coenzyme Q10, and vitamin E interfere with the lipid peroxidation by neutralizing the free radicals. In particular, liposoluble scavengers in cellular membranes have high diffusion rates, enabling them to abolish the radical chain reactions by immediately converting them into more stable and less reactive molecules (46). Additional defence mechanisms that reconstruct damaged molecules involve using specific phospholipase that removes peroxidized fatty acids, allowing the reacylation of damaged molecules (51, 52).

General overview of thyroid diseases
Thyroid hormones have a considerable impact on the cellular oxidative stress processes which is ascribed to their role in cellular metabolism and oxygen consumption (53). Thyroid hormones are produced by thyroid gland, released into circulation, and transported to all organs and cells where they exert their effect. An important role in production of thyroid hormones has hypothalamic-pituitary-thyroid axis. Hypothalamus production of thyrotropin-releasing hormone (TRH) stimulates anterior pituitary gland to secrete thyroidstimulating hormone (TSH), which affects thyroid gland and leads to production of thyroid hormones. Thyroid gland mainly produces T4, a prohormone which needs to convert to T3 to become biologically active. T4 comprises about 80% of secreted thyroid hormones, while the other 20% is T3. Increased plasma values of thyroid hormones in circulation activate negative feedback loop and inhibit release of TSH (54, 55).
Thyroid hormones exhibit profound metabolic effects characterized by an increased rate of both catabolic and anabolic reactions, resulting in an overall acceleration of the basal metabolism which is associated with increased oxygen consumption, respiratory rate, energy expenditure, and heat production (56). In addition, altered thyroid hormones levels may cause changes in the number and activity of mitochondrial respiratory chain components which represent the principal cellular site of ROS production, ultimately leading to changes in the cellular redox environment and increased ROS generation (57,58). For instance, it has been reported that hypothyroidisminduced dysfunction of the mitochondrial respiratory chain is associated with increased production of free radicals (59) Thus, excess TSH in hypothyroidism may modulate oxidative stress processes (60) by augmenting the accumulation of ROS that result from both increased generation of free radicals and diminished capacity of the antioxidative defense systems.
Hypothyroidism is associated with an increased risk of atherosclerosis due to its metabolic effects (61, 62). It is commonly accompanied by hyperlipidemia which results from a disbalance between the rates of fatty acids' synthesis and degradation and is characterized by elevated total cholesterol and low-density lipoprotein-cholesterol (LDL-C), thus providing the substrate for ROS-mediated lipid peroxidation (63-66). Interestingly, products of lipid peroxidation may further increase overall cellular oxidative stress by facilitating the generation of free radicals through the formation of adducts with proteins, which increases direct free radical-induced protein oxidation (67).
In thyroid diseases, metabolic disorders associated with lowgrade inflammation can also lead to an increased oxidative stress (68). For instance, chronic low-grade inflammation observed in Hashimoto's thyroiditis causes endothelial dysfunction which represents an early step in the development of atherosclerosis. Endothelial dysfunction is characterized by the reduction of bioavailability of NO, resulting in impaired endotheliumdependent vasodilation (69) and increased oxidative stress (70). However, it should be mentioned that there is still no consensus in the literature regarding the connection between hypothyroidism and oxidative stress. Some studies report increased oxidative stress in hypothyroidism while other suggest that hypometabolic state that is prevalent in hypothyroidism may protects tissues from oxidative damage.
Thyroid diseases are considered the most commonly reported endocrine diseases in clinical practice, followed by lipid and carbohydrate disorders (71-73). The primary thyroid condition that affects thyroid functionality presents as hyperthyroidism or hypothyroidism (Figure 1.). Thyroid dysfunction could manifest fully (clinically) or latently (subclinically). Based on duration, thyroid dysfunction could be persistent or transitional (73-75). The natural history of thyroid disorders can negatively affect the morphology and function of target tissues if left untreated or improperly treated (55, 73, 76). Thus, the leading causes of death in patients with thyroid dysfunctions are the consequences of atherosclerosis acceleration and the worsening of pre-existing cardiovascular and central nervous system diseases (77, 78). Such endpoints depend on pronounced OxS and diminished antioxidant defense systems at the molecular level (79-81).

Thyroid diseases and OxS
Different thyroidopathies have been shown to cause increased ROS production and evident OxS-induced damage to thyroid cells. This relationship is reciprocal since thyroid conditions can worsen OxS and increase ROS production, exacerbating oxidative damage. Thyroid hormones increase ROS release in the mitochondrial respiratory chain (25, 82, 83). Hypothyroidism contributes to OxS through an inefficient antioxidant defence system, opposite to hyperthyroidism, where increased ROS production promotes OxS and oxidative damage of thyroid cells ( Figure 1 Thyroid gland's response to oxidative stress in: (A) physiological and (B) pathological conditions. Biorender.com was used to generate part of the Figure. preventive dietary antioxidant therapy may partially correct the redox imbalance, making it a viable method for preventing the onset of many chronic thyroidopathies (25).

Hypothyroidism
Even in its subclinical form, hypothyroidism reduces antioxidant system activity, which promotes OxS, causing oxidative damage and altered lipid metabolism in thyroid cells (25, 46, 84). MDA, a by-product of ROS-induced lipid peroxidation, has also been found in higher serum concentrations in hypothyroid patients (79). Even though levothyroxine considerably reduces lipid peroxidation, the serum MDA levels never reach the levels seen in healthy individuals (85). In addition, accumulated oxygen free radicals in thyroid cells may inhibit TPO function and interfere with the synthesis and secretion of thyroid hormones, causing hypothyroidism (34, 86).

Hyperthyroidism
ROS generation is increased by hyperthyroidism (87). The increased intracellular ATP consumption, increased tissue oxygen consumption and oxidative phosphorylation, overexpression of adrenergic receptors, and a decrease in antioxidant defensive mechanisms are the mechanisms of free radicals overproduction that favour OxS in hyperthyroid patients (46). In addition, patients with hyperthyroidism have increased rates of lipid peroxidation compared to euthyroid people, which is a sign of oxidative damage to membrane lipids (82). The link between hyperthyroidism and deteriorating OxS is suggested by the positive association between thyroid hormones and MDA, TSH, and GSH (83).

Nodular goitre
OxS promotes thyroid cell proliferation (88, 89). Elevated MDA levels were observed in tissues collected from patients with toxic and non-toxic multinodular goitre, accompanied by reduced activity of SOD, GPx, and selenium content compared to adjacent, healthy thyroid tissue. Tissues of benign thyroid nodules show significantly reduced total antioxidant status (TAS) and reduced oxidative stress index (OSI) (90). The presence of elevated OxS parameters in toxic multinodular goitre and decreased plasma GPx and GR activities were also demonstrated (91). These findings suggest an impaired redox balance and antioxidant defence in patients with toxic thyroid nodules and nodular goitre.
Additionally, rare loss-of-function germline mutations of Kelch-like ECH-associated protein 1 (KEAP1) could be detected in nodular goitre leading to Nrf2 pathway activation that favours transcription of cytoprotective and antioxidant enzymes (92). The thyroid nodule size may change in both directions over time. The decrease in the size of thyroid nodules may result from supplementation with extracts of plants with antioxidant and anti-inflammatory properties (93).

Hashimoto thyroiditis
By interacting with TPO and thyroglobulin (TG) and promoting immunogenicity by altering their morphology and function, NADPH-oxidases (NOXs) involvement in the production of hydrogen peroxide (H 2 O 2 ) regarding thyroid hormone synthesis may be related to the pathophysiology of AITD (86, 94, 95). More specifically, it has been demonstrated that an increase in ROS encourages the cleavage of TG into smaller fragments, which exposes the immune system to novel epitopes and intensifies the autoimmune response (96). OxS indicators are significantly higher when Hashimoto thyroiditis is associated with thyroid dysfunction. According to certain studies, the markers of worsened OxS in patients with Hashimoto thyroiditis were closely related to the levels of TG or TPO antibodies (25, 97-99).
Because it increases ROS production and lowers antioxidant levels, excessive iodine consumption is considered an additional risk factor for developing AITD. In people with Hashimoto thyroiditis, anti-TPO antibodies depend on GSH levels and exhibit an inverse correlation (89, 100). Additionally, there is a favourable association between total oxidative status (TOS) and OSI and both antibodies (anti-TG and anti-TPO). Reduced GSH levels seem to be a decisive factor in OxS activation and the development of Hashimoto thyroiditis (101, 102). Additionally, it has been demonstrated that elevated TOS and OSI parameters may precede the development of hypothyroidism in autoimmune thyroiditis and may serve as indicators of thyroid cell injury (101-103). Areas with lower-selenium soil have been linked to increased Hashimoto thyroiditis in humans (104). Also, genetic interactions between minor alleles in the selenoprotein S gene (SELENOS) and the nuclear factor erythroid 2-related factor 2 gene (NFE2L2) increase chronic thyroiditis incidences (105).

Graves' disease
The most typical cause of hyperthyroidism is Graves' disease (GD). Its natural history appears to be heavily influenced by oxidative DNA damage. Untreated GD sufferers were shown to have much more DNA damage than patients with toxic nodular goitre and healthy people (106). The highest level of OxS markers was recorded in hyperthyroid GD patients, especially ones with relapsing disease (107, 108). Although both thiamazole and propylthiouracil effectively restore ROS and the antioxidative defence systems, some authors evidenced propylthiouracil as more efficacious (109). The unique mechanism of how OxS leads to GD is disrupting selftolerance. The thyroid-stimulating antibodies (TSAb) present in GD are engaged in oxidation processes. As the markers of OxS show a positive correlation with TSAb, it may indicate that these variables may be involved in the breakdown of redox balance (110). In patients with GD, activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway may help restore thyroid function (105).

Thyroid cancer
Increased production of ROS has been shown to favour cancer development (111). However, ROS can also trigger cell senescence and death, acting as an anti-tumorigenic agent (112). Disturbed genomic integrity induces oxidative genetic damage, DNA oxidation, the activation of proto-oncogenes, and the inactivation tumour suppressor genes leading to proliferative effects and mutagenesis (12,113,114). According to Krohn et al., DNA damage, a precursor to tumorigenesis, is thought to be caused by OxS (115). Resultant oxidative DNA base lesions have the potential to mutate some genetic material, which would impair the integrity of the genome by preventing transcription and replication and by generating mutagenesis (116). The oxidized form of guanine, 8-oxo-2′-deoxyguanosine (8-oxo-dG), is a valuable marker of oxidative DNA damage during carcinogenesis (116, 117). When compared to matched normal thyroid tissue, both benign (human follicular adenomas, or FTAs) and malignant (follicular (FTC) and papillary thyroid carcinoma (PTC)) lesions were found to have elevated nuclear levels of 8-oxo-dG (118) which most likely reflects the detrimental effects of prolonged exposure to chronic OxS seen during thyroid cancer (113,118).
According to an analysis of the redox balance, sera antioxidant levels were lower in thyroid cancer patients than in healthy controls, and OxS marker sera levels in thyroid cancer patients were significantly higher than in the control samples (119). In addition, high concentrations of MDA in blood were detected in thyroid cancer patients, which unequivocally indicated reduced blood antioxidative capacity (120,121).
A disturbed balance between serum OxS and antioxidant defence system markers is typically encountered in thyroid cancer patients compared to healthy individuals (119,120,122,123). The ineffective defence mechanism cannot neutralize ROS overproduction in thyroid cancer cells, leading to OxS (122). A significant difference in GPx activity and MDA levels was seen between the thyroid cancer patients before and after thyroidectomy in a study examining the change in OxS markers. Although thyroidectomy dramatically improved the oxidative status in favour of antioxidants, lipid peroxidation levels remained much more significant than in healthy thyroid people (25, 121, 124). Also, PTC patients exhibit a worse oxidative profile than patients with autoimmune thyroid disease and higher oxidative process rates than healthy individuals (125). Moreover, thyroid cancer risk was observed to be higher in obese people, and female patients with type 2 diabetes mellitus (T2DM) are more likely to have the extraglandular invasion of PTC than male T2DM patients (126,127).
In PTC and anaplastic thyroid carcinoma, somatic KEAP1 and NFE2L2 mutations activating the Nrf2 pathway were discovered (128, 129). Although the significance of such pathway activation in thyroid tumours is still unclear, it may help cancer cells survive (105, 130). In addition, tumour tissue exhibit a higher quantity of ROS, which was linked to the decreased expression of selenium antioxidant proteins in cancer cells compared to healthy cells (122). Furthermore, compared to normal thyroid tissue, antioxidant catalase expression was significantly reduced in human thyroid tumours (131). These results show oxidant/antioxidant system in thyroid cancer tissue is imbalanced (12, 132).

Nutrition and OxS
Proper nutritional intake is mandatory for overall well-being and better human health. However, dietary habits have an impact on human health and can lead to the development of a variety of disorders and diseases. The most prevalent diet in the world is the Western-style diet, characterized by an increased intake of refined food with a high caloric index and an increased amount of sugars and salt, while the intake of vegetables, fruits, and fish is reduced (133). Negative consequences of nutritional habits associated with a Western-style diet may lead to inflammation and production of free radicals (134) through the secretion of numerous pro-inflammatory molecules such as interleukin (IL)-6, IL-1b, IL-8, and C-reactive protein (CRP), leading to the development of autoimmune disorders either directly, due to inflammation or disturbed immune balance, or indirectly due to increased fat depositions and the development of obesity. Obesity has the most severe consequences since it is associated with systemic inflammation, hypertension, and hypercholesterolemia, which represent conditions that increase the risk of developing cardiovascular disease and T2DM.
It is believed that the state after taking a meal (postprandial state) is pro-inflammatory and pro-oxidative, and the type of food mainly consumed affects the occurrence of OxS. As mentioned earlier, the increased intake of proteins (processed, red meats), sugars, salt, saturated and trans fat, and refined carbohydrates, which are characteristic of the Western-style diet, leads to the development of many diseases, which have their basis in the occurrence of OxS (135). Furthermore, a diet based on an increased intake of carbohydrates and fats leads to increased production of free radicals, directly affecting mitochondrial metabolism. Also, animals fed a high-fat diet have been shown to have increased OxS and dysfunctional mitochondria (136).
To reduce the development of obesity, cancer, diabetes, and cardiovascular diseases, WHO recommended a diet that includes an increased intake of fruits, vegetables, nuts, fish, and unsaturated fatty acids. This type of diet is represented in certain coastal regions of the world and has received the popular name Mediterranean diet. The natural antioxidants, as a result of proper nutritional habits, provide indirect protection by decreasing the production of cytokines and reducing OxS (134).
Numerous exogenous antioxidant molecules (nutritional antioxidants) have been shown to play an important role in excessive ROS accumulation in organisms. Here we will discuss several nutritional antioxidants with a confirmed protective antioxidant role. For instance, monounsaturated fatty acids (MUFA) such as oleic acid that are present in high amounts in olive oil and certain nuts decrease ROS production and exert protection against OxS (137). In addition, oleic acid showed anti-inflammatory effects by decreasing obesity and cytokine production and reducing cardiovascular mortality (134). The anti-inflammatory actions of MUFAs are based on their ability to counteract the effects of long-chain saturated fatty acids on hepatocytes, which include reducing endoplasmic reticulum stress, restricting lipotoxicity induced by accumulation of saturated fatty acids, decreasing ROS production, and inhibiting nuclear factor-kB (NF-kB) transcription factors by binding peroxisome proliferator-activated receptor g (PPARg) and G-protein coupled surface receptor 120 (GPR120) (138). In vitro, MUFA has shown the ability to induce the expression of the adiponectin gene via PPARg activation, which would result in decreased production of pro-inflammatory molecules such as IL-6 and tumour necrosis factor (TNF)-alpha (139) Polyunsaturated fatty acids, such as omega-3 fatty acids (n-3 PUFA), are mostly found in eggs, nuts, and fish, whereas omega-6 fatty acid (n-6 PUFA) is predominantly present in sunflower and other vegetable oils. The ratio of n-3 PUFA/n-6 PUFA is of the utmost importance since its disbalance may activate proinflammatory pathways (140) n-3 and n-6 PUFA exert opposite effects on the immune system, whereas n-3 PUFA have an antiinflammatory effect while n-6 PUFA induces a proinflammatory action (141). The anti-inflammatory effects of n-3 PUFA are based on their ability to decrease endogenous concentrations of ROS and expression of NF-kB and promote activation of genes involved in antioxidant protection.
Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a natural polyphenol nonflavonoid compound primarily found in grapes, red wine, berries, and peanuts. It has been shown that long-term treatment with reservatrol prolongs lifespan and reduces OxS (142). In addition, resveratrol was shown to possess cardiovascular protective capacity (143-145) and exhibit antidiabetic, antiinflammatory, and antioxidant effects (146-150), as well as the ability to suppress the proliferation of a variety of tumour cells (151,152). Also, resveratrol positively affects obesity, reducing triglycerides and glucose levels. The anti-inflammatory effects resulting from using resveratrol can be seen in the reduction of increased levels of interleukin and TNF in obese mice (15). The antioxidant effects of resveratrol were confirmed in many studies. For example, resveratrol significantly inhibited ROS production by polymorphonuclear leukocytes treated with formyl methionyl leucyl phenylalanine (153) and reduced OxS markers like glycated albumin levels in serum and 8-hydroxyguanosine in urine in stroke-prone spontaneously hypersensitive rats (154). Due to its lipophilic nature, resveratrol can bind to lipoprotein particles, which seems crucial for its antioxidant effects (155). Resveratrol consumption increases plasma antioxidant levels and decreases lipid peroxidation (156). It also reduces intracellular ROS and prevents LDL oxidation in endothelial cells (157) by inhibiting lipoxygenases (158). The mechanism by which resveratrol prevents LDL oxidation is based on its ability to chelate copper and scavenge ROS (159).
It is important to mention micronutrients with antioxidant properties, such as vitamins. Since the body cannot synthesize sufficient amounts of vitamins, it is necessary to take them through food or supplements. Vitamin E acts as a regulator of cellular metabolism, and its deficiency leads to anaemia, dysregulation of energy metabolism, irregular mitochondrial function, and tissue damage resulting from increased lipid peroxidation (16). Vitamin D deficiency affects muscle function and leads to irregular cardiovascular function. Also, a lack of Vitamin D is connected with disrupted mitochondrial function and thus increased inflammation and OxS (16). However, it should be emphasized that an increased intake of vitamins has the opposite effect, increasing OxS. For example, it is well known that vitamin C has antioxidant properties; it reacts with ROS creating a product with poor reactivity that does not have detrimental effects. On the other hand, vitamin C may undergo the Fenton reaction, in which a highly reactive free radical is formed (170).
7 Nutritional antioxidants and thyroid disease 7.1 Trace elements 7

.1.1 Iodine
An average adult body contains around 15 to 20 mg of iodine located predominantly in the thyroid gland, which performs an essential role in synthesizing thyroid hormones. In addition to being a component of the thyroid hormone, iodine can act as an antioxidant and antiproliferative agent (171). Its uptake is mediated by the sodium/iodide symporter (NIS), expressed in thyroid cells and extrathyroidal tissues, including the stomach and salivary glands. The iodine content in food is determined by its amount in the soil. Since seafood and seaweed are rich sources of iodine, a diet based on high seafood consumption is sufficient. Likewise, fortifying salt and milk products with iodine ensures an adequate amount of dietary iodine (172,173). Although iodine deficiency was associated with goitre and thyroid nodules, PTC appears to be more common in areas with high iodine intake, which points to the complex relationship between iodine intake and thyroid disease (174). For instance, excessive iodine intake is associated with a transient reduction of thyroid hormone synthesis for approximately 24 hours after ingestion, known as the Wolff-Chaikoff effect (175). In patients with autoimmune thyroid disease or on anti-thyroid drug therapy, increased iodine intake can induce hypothyroidism, whereas, in patients with diffuse nodular goitre or latent Grave's disease, it can cause hyperthyroidism (176).

Zinc
Zinc is regarded as an antioxidative trace element because it is a co-factor of the enzyme SOD, which scavenges free radicals. Zinc is essential for normal thyroid function since it is required for the activity of enzyme 1,5′-deiodinase which catalyzes the conversion of T4 to T3. In addition, zinc plays a vital role in the thyroid hormones' metabolism by regulating thyrotropinreleasing hormone (TRH) and TSH synthesis and modulating the structures of essential transcription factors involved in synthesizing thyroid hormones (177,178). Zinc deficiency affects the thyroid gland by impairing TRH, TSH, T3, and T4 synthesis. In animal studies, free levels of T3 and T4 were reduced by approximately 30% (179), and a similar trend was observed in studies of human subjects. Hypothyroid patients often present with reduced levels of zinc. In a study designed to evaluate zinc metabolism in patients with thyroid disease, plasma and erythrocyte zinc concentration and urinary zinc excretion were investigated in hypo-and hyperthyroid patients (180). The mean concentration of plasma zinc in hypothyroid patients was lower than that of healthy control subjects, whereas no statistically significant differences were observed in plasma zinc values between hyperthyroid patients and control subjects. However, erythrocyte zinc concentration was significantly decreased in hyperthyroid patients compared to hypothyroid patients and accompanied by an increased urinary zinc excretion resulting from increased muscle tissue catabolism in hyperthyroid patients. The findings of this study suggest that abnormal zinc metabolism commonly occurs in thyroid dysfunctions (180) ( Table 1).

Selenium
Selenium is an essential trace mineral whose functions in the organism are mainly connected to its antioxidant properties (190). Selenium is essential to antioxidant enzymes such as GPx (183) and is involved in thyroid and immune system functions. The thyroid gland has the highest concentration of selenium in the body, which is predominantly stored in the thyrocytes in the form of selenoproteins, such as deiodinases, GPx, and thioredoxin reductases (181,182). Adequate selenium intake is mandatory for the normal function of thyrocytes, and selenium deficiency is associated with the decreased synthesis of thyroid hormones (191), increased thyroid volume, and increased number of thyroid nodules (182,192). Selenium has been shown to affect T-cell differentiation and modulate the Thelper (Th) cells' responses. Th cells are cytokine-producing cells that are divided into subgroups 1 and 2 depending on their mechanisms of action; Th1 cells are involved in cell-mediated immunity, whereas Th2 cells participate in antibody-mediated immunity. Th1 cytokine production generally tends to exert proinflammatory effects and may lead to autoimmune conditions such as Hashimoto's thyroiditis. Th2-induced hyperproduction of the thyroid autoantibodies observed in In Graves' disease results in hyperthyroidism. Selenium deficiency has been associated with Th2 cell response, whereas higher selenium levels favour Th1 response (193). These findings may explain the beneficial effects of selenium supplementation in autoimmune thyroid diseases (181,194), such as reduced levels of anti-thyroid antibodies, improved thyroid structure and metabolism, and ameliorated clinical symptoms (181,195). Dietary forms of selenium include selenomethionine present in plant products and inorganic selenium forms used for supplementation (196). No indication of an increased risk of thyroid cancer in either selenium deficiency or exogenous supplementation has been reported (182) ( Table 1).

Natural polyphenols and alkaloids
As an antioxidant polyphenolic compound and a free radical scavenger, resveratrol has attracted interest for the potential treatment of thyroid diseases accompanied by increased ROS production, such as autoimmune thyroid itis and hyperthyroidism (197). In addition, resveratrol may help treat thyroid cancer since it can induce apoptosis of thyroid cancer cells by increasing the abundance and phosphorylation of p53 tumour suppressor protein (p53) (198,199). In vitro and in vivo studies have also demonstrated that resveratrol mediates the levels of TSH and iodide uptake in thyrocytes by decreasing NIS expression (184). However, the observed effects also resulted in significant proliferative action of thyrocytes; thus, resveratrol may be a thyroid-disrupting compound and a goitrogen (184). Currently, data from clinical studies on resveratrol's effect on the thyroid in humans are absent, and all literature evidence is based on studies performed in cell cultures and animal models. Therefore, proper randomized clinical trials are mandatory to reach the final verdict on the potential use of resveratrol in treating thyroid diseases.
Alkaloid antioxidant berberine was recently reported to exert positive effects in treating GD. When supplemented in combination with methimazole, berberine significantly altered the microbiota composition of patients, decreasing the abundance of the pathogenic bacteria Chryseobacterium indologenes and Enterobacter hormaechei while simultaneously increasing the content of the beneficial bacteria Lactococcus lactis (185). In addition, berberine supplementation resulted in significantly elevated enterobactin production, improving iron functioning and restoring thyroid function in patients with Graves' disease (185) (see Table 1).

Inositol
Inositol (also known as vitamin B8) is a carbohydrate compound that is an essential component of the plasma membrane phospholipids and has an important role in synthesizing secondary messengers in the cells (200). Inositol is involved in signaling hormones such as TSH, insulin, and gonadotropins. Myoinositol (Myo), a cyclic polyol with six hydroxyl groups, is the most abundant isoform of inositol, mainly derived from the dietary intake of fruits, beans, and nuts. In contrast, its endogenous production is generated either from glucose by enzymatic reactions or by de novo catabolism of phosphatidylinositol (PI), phosphoinositides (PIP), and inositol phosphates (IP). Myo has a crucial role in thyroid function and autoimmune diseases due to its regulation of thyroid hormone synthesis by forming H 2 O 2 in thyrocytes. Myo is involved in the TSH signaling pathway; thus, depleted levels of Myo may cause the pathogenesis of thyroid diseases such as hypothyroidism (187). It has been observed that TSH levels significantly decreased in patients with subclinical hypothyroidism, with or without autoimmune thyroiditis, after treatment with Myo in combination with selenium (201,202). Studies of patients with Hashimoto's thyroiditis and subclinical hypothyroidism showed that supplementation of Myo and selenomethionine significantly decreased TSH, TPOAb, and TGAb concentrations, while simultaneously increasing thyroid hormones levels and restoring euthyroid state in patients with autoimmune thyroiditis (203,204). In addition, the combined treatment with Myo and selenomethionine was found to have an ameliorating effect on nodular thyroid disease by promoting a significant reduction of thyroid nodules size and number and regression of their stiffness (205). Additional in vitro and in vivo studies are required to investigate the mechanism of this effect and the potential use of Myo, alone or in combination with selenomethionine, as a novel clinical treatment for the general management of autoimmune thyroiditis and thyroid nodules ( Table 1).

L-carnitine
L-Carnitine (3-Hydroxy-4-(trimethylazaniumyl) butanoate) is a biological compound that is ubiquitous in mammalian tissues and fluids where it is required for b-oxidation of fatty acids by facilitating their transport in the form of acyl-carnitine esters across the mitochondrial inner membrane (206). In addition, L-carnitine possesses significant antioxidant properties reflected in its ability to scavenge superoxide anion radical and hydrogen peroxide and chelate metal ions such as ferrous ions (207). L-carnitine was shown to positively impact cardiac function through reduced oxidative stress, inflammation, and necrosis of cardiac myocytes (208). As much as 75% of Lcarnitine comes from the dietary intake of red meat and dairy products ( Table 2), whereas only 25% is generated by endogenous biosynthesis. Muscles are the main reservoir of carnitine, storing 95% of the total amount of 120 mmol present in the adult human body (209) The anti-thyroid effect of L-carnitine is based on its ability to inhibit thyroid hormone entry into the nucleus of hepatocytes, neurons, and fibroblasts (186, 210). As a result, rather than being

Biological compounds
Carnitine Red meat, dairy products a direct inhibitor of thyroid gland function, it acts as a peripheral antagonist of thyroid hormone action (186). The first controlled clinical trial demonstrating the beneficial effects of L-carnitine in reducing elevated thyroid hormone circulating levels was conducted in 50 women receiving TSH-suppressive (L-T4) therapy for cytologically benign thyroid nodules (210). Lcarnitine supplementation was shown to be effective in reversing and preventing symptoms of hyperthyroidism (210). Consequent studies showed that severe forms of GD-related hyperthyroidism, including thyroid storms, may be effectively treated with L-carnitine (211)(212)(213), which may be partly explained by increased levels of thyroid hormones deplete the tissue deposits of L-carnitine (214). Interestingly, decreased concentration of L-carnitine was also found in the skeletal muscles of hypothyroid patients (215), suggesting that Lcarnitine depletion in skeletal muscles may contribute to myopathy associated with either hypothyroidism or hyperthyroidism. A recent study demonstrated that L-carnitine supplementation might alleviate fatigue symptoms in hypothyroid patients (216). Further clinical studies are required to establish the usefulness of L-carnitine supplementation in hypothyroidism (see Table 1).

Probiotics
Probiotics are live non-pathogenic microorganisms with beneficial health effects for their hosts (217). Probiotics regulate the composition of the intestinal microbiota, stimulate humoral and cellular immunity; decrease the frequency and duration of diarrhoea; and eliminate harmful metabolites in the colon, such as ammonium and procancerogenic enzymes. In addition, certain probiotic strains possess antioxidant activity and may reduce damage caused by OxS (218). Probiotics improve metabolic diseases such as obesity and diabetes by modulating intestinal microbiota composition (219)(220)(221). Furthermore, the oxidative stress in patients with T2DM can be ameliorated by multispecies probiotics (222).
Probiotic bacteria possess their antioxidant defence systems, such as enzymes SOD and Cat, and can chelate metal ions, such as ferrous and cupric ions, preventing them from catalyzing oxidation (19,22,223). In addition, probiotics can stimulate the host's antioxidant defence systems and increase the activity of antioxidant enzymes (224). For instance, intact cells and cell-free extracts of Bifidobacterium animalis 01 can scavenge hydroxyl radicals and superoxide anion in vitro, whereas in vivo, they increase the antioxidative enzyme activity (20). Lactic acid bacteria strains can defend against peroxide radicals, superoxide anions, and hydroxyl radicals (225,226). Human studies have shown elevated SOD and GPx activities, and improved total antioxidant status in T2DM patients supplemented with Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 (227). Lactobacillus rhamnosus supplementation was shown to exert significant antioxidant protection in conditions of increased physical stress (228). In addition, probiotics produce various antioxidant metabolites, such as GSH and folate. Lactobacillus fermentum strains, E-3 and E-18, contain very high levels of GSH (226), which can, together with selenium-dependent GPx, eliminate hydroxyl radicals and peroxynitrite (229).
The enormous complexity of human microbiota is reflected in the finding that adult human organisms typically contain 10 14 bacteria in the gut, which is approximately ten times more bacterial cells than the number of human cells (230), with at least 400 different bacterial species (231). Most bacterial species in a healthy human microbiota belong to the genera Bacteroidetes and Firmicutes (232), whereas Actinobacteria, Proteobacteria, Fusobacteria, and Cyanobacteria, are less abundant (233). Furthermore, microbiota composition varies depending on its localization in the gastrointestinal system; therefore Bacilli class of the Firmicutes and Actinobacteria is enriched in the small intestine, whereas the Bacteroidetes family of the Firmicutes is predominantly present in the colon (234).
Gut microbiota in patients with thyroid diseases has a different composition compared to the healthy controls and typically contains a decreased content of Lactobacillaceae and Bifidobacteriaceae. (23). The family Lactobacillacae has important antioxidant properties and may exert protective effects on the thyroid. (235), and its decreased content may cause higher oxidative stress in the thyroid (23). In addition, opportunistic pathogens in gut microbiota were shown in patients suffering from thyroid disease (Zhang,235). Gut microbiota dysbiosis negatively affects the regulation of antiinflammatory and immune system responses and appears to be associated with autoimmune diseases, inflammation, and some types of cancer (189,236,237). For instance, thyroid cancer is associated with the increased presence of Clostridiaceae, Neisseria, and Streptococcus, whereas in patients with thyroid nodules, a relative increase of Streptococcus and Neisseria compared to healthy controls was observed (235). Increased abundance of Neisseria has been linked to inflammatory disorders (238). In contrast, Clostridiaceae and Streptococcus were associated with carcinogenic effects and a higher risk of carcinomas (239, 240), and those three seem to have a role in thyroid carcinogenesis (23).
Probiotic supplementation has substantial beneficial effects on thyroid hormones and thyroid function. It was demonstrated that Lactobacillus reuteri supplementation improves thyroid function in mice by increasing free T4 and thyroid mass (241). Microbiota modulation by probiotic supplementation of Lactobacilli and Bifidobacteriaceae increased levothyroxine availability in humans and stabilized thyroid function. Probiotics were shown to be beneficial in lowering serum hormone fluctuations (188), partly because iodothyronines deconjugation is regulated by bacterial enzymes sulfatases and ß-glucuronidases whose availability could be increased by probiotic supplementation (189). Finally, probiotics influence the uptake of minerals relevant to thyroid function, including selenium, iodine, iron, and zinc, and a synergistic effect of probiotics and trace elements on the total antioxidant capacity was observed in vivo. Although probiotic supplementation shows promising potential in improving thyroid function in thyroid diseases, further human studies on the effects of probiotics as adjuvant therapy for thyroid diseases are required (Table 1).

Conclusions
Besides the impairment of cellular redox homeostasis in thyroid gland cells, thyroid diseases significantly contribute to systemic redox imbalance. In that way, thyroid diseases are organ-confined and promote histological changes in distant organs by disturbing the vital cellular pathways. Therefore, the simultaneous treatment of thyroid diseases and substituting of nutraceutical antioxidants could beneficially affect different molecular mechanisms enabling the recovery of disturbed redox balance. Se levels are lower in people with thyroid dysfunctions, such as subclinical or overt hypothyroidism (242). In order to determine whether Se supplementation may impact the progression of autoimmune thyroid disease, some trials carried out in regions where the population has a diffusely low or borderline Se status inconsistently suggest that Se supplementation may cause a decrease in thyroid autoantibodies (243,244). The population heterogeneity, various Se formulations and the length of Se supplementation, as well as different thyroid function test and Se measurement strategies, are among the reasons for the study conclusions inconsistency (244). The benefit of Se supplementation could be expected in patients living in regions with low Se availability or who have low-or sub-optimal Se levels. The supplementation must be attentive as the reference ranges of Se blood levels are narrow, and the risk of insufficient or toxic supplementation is possible (245,246).
Accumulating evidence support the existence of a thyroidgut axis and displays important correlations between the composition of the gut bacteria and thyroid function. Dysbiosis, a common finding in thyroid disorders, not only promotes local inflammation of the intestinal membrane but also directly affects thyroid hormone levels via its own deiodinase activity and TSH inhibition. In addition, gut microbiota can modulate the absorption of trace minerals, such as iodine, selenium, and zinc, that are essential for thyroid function, including iron. For instance, iodine deficiency may lead to goiter, whereas high iodine intake may induce thyroid dysfunction in susceptible patients. Supplementation with antioxidative probiotics has shown beneficial effects in thyroid diseases thus representing a potential adjuvant therapy for thyroid disorders. The advances in the field of microbiome research envision the future possibility of personalized treatment with probiotics that are specifically adjusted to individual patients. Nevertheless, more data from adequately powered human studies are required for further evaluation of the impact of gut microbiota on thyroid diseases and the potential for possible therapeutic interventions.

Author contributions
MM wrote the article. ZG wrote the article. SZ wrote the article. ME wrote the article, XG wrote and critically reviewed the article and EI wrote and critically reviewed the article. All authors contributed to the article and approved the submitted version.