The interplay between vitamin C and thyroid

Abstract Introduction Vitamin C (ascorbic acid) is a water‐soluble vitamin, that plays a key role in the prevention and treatment of scurvy. As vitamin C is an antioxidant and thyroid function may be affected and may affect vitamin C levels, for the first time, we aimed to provide a detailed review of all human studies evaluating the different roles of vitamin C in the thyroid gland. Thyroid cancers, goitre, Graves' disease and other causes of hyperthyroidism and hypothyroidism were the conditions discussed in this study. Furthermore, vitamin C addition to other medications such as levothyroxine was also reviewed. Methods In this study, we reviewed the relevant literature regarding the association between vitamin C and thyroid diseases using original studies from PubMed, Scopus, Embase, and Web of Science. Results In this review, we found anti‐cancer effects for intravenous (IV) administration of vitamin C in addition to the beneficial effects of using it in combination with radiotherapy and chemotherapy. As autoimmune diseases affect some antioxidant markers, some studies reported a significant difference in blood vitamin C levels in patients with autoimmune thyroid diseases such as Graves' disease. Despite many studies evaluating the effects of IV administration of vitamin C in mentioned diseases, there is a lack of evidence for oral consumption of vitamin C. Conclusions To conclude, there is a lack of evidence, especially clinical trials, for the therapeutic effects of vitamin C on thyroid diseases; however, promising results were reported in some studies in the literature.


| INTRODUC TI ON
Vitamin C is involved in the maintenance of several body functions, and its role has been shown in several organs and systems. 1 Although its main form in the body is ascorbate, it acts as a co-substrate for several enzymes and antioxidants. 2,3 Its role as an antioxidant is well known and it is well known as reactive oxygen species (ROS) scavenger in neurons. 4,5 Its concentration in body fluids and tissues depends on intestinal absorption, cellular transport and excretion. 6 The thyroid gland, as one of the main sources of controlling metabolism and growth, plays an important role in body function, mainly through its main hormones, thyroxine and triiodothyronine. 7 Several studies have assessed the levels of vitamin C in thyroid disorders or the effects of vitamin C on the absorption of thyroid medications. However, the effect of vitamin C on thyroid function and hormones is still unknown. Herein, by reviewing the available literature, we aimed to assess the association of vitamin C on the thyroid function gland, whether as a supplement or as a peripheral biomarker by comparing its levels among patients and controls. The search terms associated with our findings are shown in Table S1. The overall findings can be categorized into the following sections: (1) thyroid cancer, (2) goitre, (3) thyroid autoimmune disorders including Graves' disease and autoimmune thyroiditis, (4) role of vitamin C in levothyroxine absorption, (5) oxidative stress, vitamin C, and hypothyroidism, (6) vitamin C effects on oxidative stress of hyperthyroid patients, (7) benign thyroid disease and (8) thyroid lesions.

| C AN CER
A growing body of evidence documented the anti-cancer potential of vitamin C, 8,9 and several pre-clinical and clinical studies have confirmed this concept. The anti-cancer effect of vitamin C was first introduced nearly 50 years ago by Pauling and Cameron. 10 They showed in clinical studies that intravenous vitamin C (∼10 g daily) could increase the survival duration of patients with incurable cancer. Vitamin C was used as oral or intravenous administration and single or combined treatment; therefore, controversial findings were observed.

| Anti-cancer mechanism of vitamin C
Several mechanisms have been proposed for the cytotoxic effect of vitamin C in cancerous cells. Different studies have shown that vitamin C acts as a multi-organ targeting agent and can play a role in epigenetic level, regulation of kinase activity, inhibition of epithelialto-mesenchymal transition (EMT), immunoregulatory effect, increasing oxygen sensation, pro-oxidant activity, etc. [11][12][13][14][15] Pro-oxidant activity is the main mechanism determined for the anti-cancer activity of vitamin C and acts on redox imbalance in a dose-dependent manner. 16 The pro-oxidant activity of vitamin C is mediated by inducing injury to deoxyribonucleic acid (DNA) molecules, inducing DNA mutation and genome instability. 17 It is recognized that catalase activity was absent in cancerous cells that make them susceptible to oxidative stress regardless of the type of cancer. 18 A high level of glucose transporter 1 (GLUT1) expression was observed in cancer cells that mediate uptake of vitamin C. Consequently, low levels of intracellular vitamin C mediate the reduction of antioxidants like nicotinamide adenine dinucleotide phosphate (NADPH) and superoxide dismutase (SOD) enzyme. Therefore, high doses of vitamin C can increase ROS levels in cancer cells and subsequently induce DNA, protein and lipid damage to cancerous cells. BRAF (v-raf murine sarcoma viral oncogene homologue B1) mutation is the most frequent gene mutation responsible for the development of thyroid cancer and induces invasion of thyroid cancer. BRAF mutation showed their antitumour effects medicated by mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ ERK) signalling. 19 The anti-cancer effect of vitamin C is shown to be medicated by directing BRAF mutation or even regardless of targeting BRAF mutation. Regarding targeting BRAF mutant thyroid cancer cells, it was shown that vitamin C plays its antitumour effect through inhibition of MAPK/ERK and PI3K/AKT pathway which was mediated by ROS-dependent manner. 20 Alongside, PLX4032 is an antitumour treatment option that inhibits BRAFV600 kinase. It was shown that vitamin C can play a role in enhancing the antitumour effect of PLX4032 by significantly increasing mitigation of MAPK/ERK as well as PI3K/AKT pathway. 21 Moreover, another study showed that the induction of ROS production and decreasing antioxidant barrier of thyroid cancer cells by vitamin C selectively occurred in BRAF-mutated cells. 22 Redox homeostasis is responsible for several cellular mechanisms such as reaction to ROS and oxidationreduction reaction. In this regard, it was shown that vitamin C has also an impact on redox haemostasis and subsequently on nicotinamide adenine dinucleotide (NAD) salvage mechanism and tricarboxylic acid (TCA) cycle which eventuate cell death. 22 Vitamin C can also increase the apoptosis of cancerous cells by inhibiting B-cell lymphoma-2 (BCL-2) expression and increasing the expression of BAX and caspase-3 which results in cell apoptosis. 23 Additionally, vitamin C can decrease hypoxia-inducible factor-1 (HIF-1) which is essential for the endurance of cancerous cells in the hypoxia which increase the vulnerability of cancer cells to hypoxic condition. 24 Moreover, vitamin C can bear therapeutic potential for cancer treatment through the activation of tumour suppressor genes like p53 and p21 which are associated with cell cycle arrest and inhibition of cancer proliferation. 23 Furthermore, it was shown that vitamin C can play a role as a carcinostatic agent by inhibiting angiogenesis via decreasing the expression of angiogenesis-related genes such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and matrix metallopeptidase 2 (MMP2). 25 Anaplastic thyroid cancer (ATC) is a rare type of thyroid cancer that is associated with poor prognosis and there is no evidence of a definite treatment option for the management of ATC. 26 It was shown that vitamin C can significantly decrease the proliferation and advancement of ATC cells by activation of ferroptosis and irondependent lipid peroxidation. Vitamin C has been shown to have an effect on ferritinophagy that subsequently resulted in the degeneration of ferritin and discharge of free iron. Discharged free iron can subsequently stimulate ROS generation mediated by the Fenton reaction. The ROS reaction and lipid-peroxidation induced by free iron can eventuate to ferroptosis and elimination of ATC cells. 27 The effect of vitamin C is not limited to the impacts on cancerous cells. It was also documented that vitamin C can also stimulate the immune cells' reaction and can activate natural killer cells, T cells and monocyte that are responsible for the immune system in the fight against cancers. 28,29 The thyroid hormone plays a critical role in regulating branched-chain amino acid (BCAA) metabolism by modulating the expression of branched-chain aminotransferase (BCAT) and the activity of regulatory kinases and phosphatases involved in BCAA uptake. Figure 1 illustrates the key mechanisms involved in thyroid hormone-mediated regulation of BCAA metabolism, highlighting the importance of understanding these pathways for developing targeted therapies for metabolic disorders and thyroid-related diseases.
Moreover, vitamin C plays a crucial role in regulating iron metabolism by promoting ferroportin-mediated iron export and inhibiting hepcidin expression. Additionally, ferritinophagy, the autophagic degradation of ferritin, is an important mechanism for maintaining iron homeostasis and preventing oxidative stress caused by excess iron. Figure 2 illustrates the interplay between vitamin C, ferritinophagy and ROS in regulating iron metabolism.

| Pre-clinical studies
Pre-clinical studies have shown that a millimolar range of vitamin C is required for destroying cancerous cells that are not reachable through oral administration, and intravenous infusion is the optimal route of administration. 30,31 Several pre-clinical studies have investigated the effect of high doses of vitamin C on challenging tumours such as glioblastoma multiform (GBM), breast cancer, colorectal cancer, mesothelioma and pancreatic cancer and showed promising results on the anti-cancer effect of vitamin C through hampering the tumour progression and tumour metastasis. 8,16 Applying high doses of vitamin C in KRAS gene mutation-driven tumours has resulted in progression in pre-clinical studies of vitamin C. It was shown that vitamin C directs mitochondrial membrane and metabolic components and reduces the levels of adenosine triphosphate (ATP) and GLUT1, which can also make KRAS mutant cells vulnerable to chemotherapy agents. 16 Therefore, high doses of vitamin C opened up a new way to treat cancer and can be considered adjuvant therapy with other therapeutic options. Besides, vitamin C can also present as a protective treatment against the complications of other therapeutic agents meanwhile employed as a combination therapy. 25

| Clinical studies
In light of the anti-cancer potential of vitamin C in cancer management, Phases I and II clinical trials reported favourable signatures of the safety and efficacy of vitamin C as combination therapy or even monotherapy in the treatment of various cancers. 9,32 Clinical studies of vitamin C monotherapy in terminal cancer showed that a high dose of vitamin C, up to 3 g/kg, has no considerable safety issues. 30 In line with pre-clinical studies, several clinical studies have documented that vitamin C can provide long-term survival for patients with cancer, even with terminal or metastatic cancer. 16 However, no clinical studies have investigated the efficacy of vitamin C monotherapy in patients with late-stage cancer without preceding F I G U R E 1 Transcriptional and hormonal regulators of BCAA catabolising enzymes and its association with thyroid hormone. treatment. The key difference in the outcomes is thought to be related to the route of administration and pharmacokinetics of vitamin C. 31 Hoffer et al. 33 designed a clinical trial study on end-stage cancer and showed that no patient had a response to ascorbic acid.
Besides, it was also demonstrated that high-dose oral administration of vitamin C is successful in reducing the risk of development of gastrointestinal cancers, cervix, colorectal and breast cancer. 34,35 Regarding oral administration of vitamin C, other studies revealed that daily intake of vitamin C is increased the survival of patients with breast cancer, 36,37 which implies that vitamin C has a clinically beneficial antitumour effect regardless of the route of administration. Even though high-dose intravenous administration of vitamin C is associated with alternative medical agents in cancer treatment, there is a lack of sufficient evidence regarding the clinical efficacy of the antitumour effect of vitamin C. 38 Therefore, it is essential to design further clinical studies addressing the clinical efficacy of the anticancer effect of vitamin C to set up appropriate evidence for the clinical efficacy of vitamin C in practical guidelines.

Cancers can influence patients' quality of life, and vitamin C is
shown to positively affect pain relief and well-being. 39,40 Moreover, vitamin deficiency is a common condition in patients with cancer, and anti-neoplastic syndrome medications can also improve vitamin C deficiency and well-being. 41 Constitutional manifestations such as fatigue, depression, nausea, pain and loss of appetite are common in patients with cancer, and it was shown that intravenous administration of vitamin C reduces these complications and has palliative applications. 40,42 Altogether, high-dose vitamin C was shown to prolong the survival duration of patients, better performances and less pain compared to the control patients without receiving vitamin C. 42

| Combination therapy
The effect of vitamin C as a combination therapy with other therapeutic options like chemotherapy, radiotherapy, targeted therapies and immunotherapy has also been studied. 43 It was shown that vitamin C could enhance the efficacy of monotherapies agents like cisplatin, 23  anti-cancer effect. 43 Other studies have also investigated the efficacy of combination therapy of vitamin C with targeted therapies.
Vitamin C can be used as a combination therapy with radiotherapy. However, it is well known that radiopharmaceutical agents like Iodine-131 have mistaken toxicity on human lymphocytes through interruption of the double-strand breaks (DSBs) repairing system and increasing the level of DSB that results in cell death F I G U R E 2 Anaplastic thyroid cancer cells mechanism in which vitamin C activates ferritinophagy to induce ferroptosis. and malignancy. 48 Despite this, it was investigated that vitamins like vitamin E and vitamin C as antioxidant agents are game changers and can reduce the toxicity level of radiopharmaceuticals with a higher efficacy of vitamin C. 49,50 Clinical studies on the combination therapy of vitamin C with other therapeutic approaches demonstrated no obvious safety issues related to ascorbic acid.
In two clinical trials of combination therapy of ascorbic acid and gemcitabine, the patients' toxicity was attributed to the gemcitabine not related to vitamin C. 45,46 Studies documented that vitamin C has a synergic effect with kinase inhibitors like sorafenib, 47 cetuximab 13 and gefitinib 12 which can also have related to eliminating drug resistance to these agents.
Moreover, few studies have investigated the combination therapy of vitamin C and immunotherapy. It was shown that high-dose vitamin C could enhance the immunogenicity of effector T cells nor regulatory T cells and have synergic effects with immune checkpoint inhibitors that significantly increased the immunogenicity. 51,52 In addition to optimising cancer therapies, vitamin C can reduce the complains related of chemotherapy and radiotherapy, enhance the quality-of-life of patients with cancers and have protective effects on the glands. 41,53

| Vitamin C in thyroid cancers
Studies have shown that vitamin C has potential as an anti-cancer agent in the treatment of thyroid cancer. Vitamin C has been found to sensitize BRAF V600E thyroid cancer to PLX4032, a targeted therapy, by relieving the feedback activation of MAPK/ERK and PI3K/AKT pathways. 21 Mechanistic studies have also revealed that vitamin C inhibits the MAPK/ERK and PI3K/AKT signalling pathways in BRAF wild-type or mutant thyroid cancer cells. 20 Vitamin C has also been found to inhibit the growth of papillary thyroid carcinoma (PTC) cells. 22 In addition, high-dose intravenous vitamin C has been shown to be a promising multi-targeting anticancer agent in eradicating tumour cells of various cancer types, including thyroid cancer. Vitamin C induces ferroptosis in anaplastic thyroid cancer cells, which suggests its potential as a therapeutic agent. 16 Vitamin C inhibits the MAPK/ERK and PI3K/AKT signalling pathways in thyroid cancer cells through a ROS-dependent mechanism. 16,20 The data from these studies demonstrate that vitamin C kills thyroid cancer cells by inhibiting these pathways via distinct mechanisms. 54 In addition, vitamin C ROS dependently inhibits the activity of MAPK/ERK signalling via distinct mechanisms between ATP levels in BRAF mutant and wild-type thyroid cancer cells.
Overall, the exact mechanisms by which vitamin C inhibits these pathways in thyroid cancer cells are not fully understood and require further investigation. 20 Several studies evaluated the role of vitamin C in thyroid cancer as a protective agent against cancer, a radioprotective agent and its therapeutic potential. and one in patients with APC. 27 Other studies used patients with all types of thyroid cancers in their study. Cheng et al. 55 found that vitamin C increased the uptake index and excretion of parotid glands in patients with DTC. In line with the previous study, Tong et al. 53 found that Vitamin C improved the secretory function of the parotid gland in patients with DTC who underwent radioiodine therapy. In contrast, Liu et al. 59 found no benefits in adding vitamin C as vitamin C had no effect on the salivary absorbed dose of radioiodine at any time after its administration in patients with thyroid cancer. Two studies found radioprotective effects in patients receiving radioiodine therapy. 56,57 Another study in patients with DTC found that vitamin C (especially in combination with amifostine) can reduce the side effects of radioiodine therapy. 50 Two studies found a potential therapeutic role for vitamin C in thyroid cancer patients with BRAF V600 E mutation as vitamin C has antitumour effects and can kill cancer cells by MAPK/ERK and PI3K/ AKT pathways. In a recent study by Wang et al. 27 supplementation with vitamin C supplementation led to ferritinophagy, leading to the release of free iron and the released iron rejiggered ROS production resulting in ferroptosis of ATC cells. Davanzo et al. 60 found protection against thyroid cancer in patients receiving vitamin C; however, O'Grady et al. 61 found an increased risk of thyroid cancer in patients with higher vitamin C intake.

| G OITRE
Goitre is an enlarged thyroid gland due to several causes including but not limited to autoimmune disease, iron deficiency or thyroid nodules. 62 Surgery is not the only treatment plan for all patients; however, for symptomatic moderate to large goitre and failure of medical treatment, surgery is the choice. The roles of vitamin C in goitre were described in several ways. Özdem et al. 63

| THYROID AUTOIMMUNE DISORDER S
Vitamin C has been shown to be involved in cellular functions of both innate and adaptive immune systems. Its antioxidant effects as a cofactor for numerous biosynthetic and gene regulatory enzymes play important roles in several immune-modulating ways. These include neutrophil migration to the infection site, phagocytosis enhancement and generation of oxidants and also microbial killing. In this section, we aim to investigate the role of vitamin C in two autoimmune disorders of the thyroid such as Graves' disease and autoimmune thyroiditis.

| Graves' disease
Graves' disease, the most common cause of hyperthyroidism, is an autoimmune disease affecting the thyroid gland. 66  Another study by Londzin-Olesik et al. 68 in patients with Graves' disease and active thyroid-associated orbitopathy, investigated the impact of thyroid hormone levels on selected antioxidant markers.
Patients were divided into hyperthyroid and euthyroid based on thyroid hormone levels. In addition, 20 age-and sex-matched controls were also involved. Vitamin C levels were significantly lower in hyperthyroid compared to healthy controls; however, this difference was insignificant compared to euthyroid patients. Authors suggested this decrease in vitamin C levels to excessive use and increasing demand in hyperthyroid patients with Graves' disease. In patients with Graves' disease and active orbitopathy, Londzin-Olesik et al. 69 found decreasing levels of vitamin C after systemic intravenous and oral methylprednisolone compared to 20 healthy age-and TA B L E 1 Studies evaluating the association between vitamin C in patients with thyroid cancer.

References
Year

| Autoimmune thyroiditis
In a study conducted by Taddei et al. 70  suggested that the mechanism is endothelial dysfunction by oxidative stress production by cyclooxygenase activity. [71][72][73] Only one randomized controlled trial by Karimi et al. 74  The impact could be to the extent that, in some case reports, parenteral levothyroxine was administered without a clear reason for the underlying pathophysiological malabsorption mechanism. 75

| VITAMIN C EFFEC TS ON OXIDATIVE S TRE SS OF HYPERTHYROID PATIENTS
The impact of vitamin C supplementation on oxidative stress was investigated in hyperthyroid patients treated with propylthiouracil in the study by Seven et al. 85 In this analytical study, supplemental ascorbic acid of 1000 mg/day was given to hyperthyroid patients and healthy controls. Several oxidative factors were measured, and it was found that vitamin C consumption was statistically associated with an increased glutathione concentration and glutathione peroxidase activity, while it was related to the reduction in the glutathione reductase and Cu/Zn superoxide dismutase activities. These observations suggest relief in oxidative stress in this population. In another study conducted in Turkey, hyperthyroid patients were assessed and compared with healthy controls for antioxidant levels. 82 The authors showed that there was no significant difference between hyperthyroid patients and healthy

| B ENI G N THYROID D IS E A S E
Moncayo et al. 86 investigated the role of vitamin C in benign thyroid diseases. Subjects were divided into three categories: (1) immunogenic thyroid disease (n = 112), (2) subacute thyroiditis (n = 29) and (3) normal thyroid (n = 273). Levels of vitamin C were higher in blood samples of patients with subacute thyroiditis compared to immunogenic thyroid disease (6.48 ± 3.12 μg/L vs. 6.10 ± 2.63 μg/L). In addition, both abnormal thyroids populations had higher concentrations of vitamin C in comparison with healthy controls (6.07 ± 2.94 μg/L in normal thyroid). In addition, they found that low levels of vitamin C affect Se action and metabolism in benign thyroid disease patients.

| THYROID LE S IONS
In a research by Jóźwiak et al. 87 the association between the expression of HIF-1α and HIF-2α and levels of vitamin C in thyroid lesions was studied. Researchers used thyroid lesions from 106 nodular thyroid disease patients who underwent surgical resection. They found an inverse relation between tissue ascorbate level and HIF-1α expression (r = −0.288, p = .025). In addition, no difference between vitamin C levels of thyroid lesions was detected. The findings of this study suggest that intracellular vitamin C levels in thyroid lesions are not different and extracellular accumulation of vitamin C changes in thyroid lesions.

| CON CLUS ION
Vitamin C, or L-ascorbic acid, is an essential vitamin with antioxidant properties, which is essential for both preventing and treating different diseases. We intended to offer a thorough overview of all human studies investigating the various roles of vitamin C in the thyroid gland because vitamin C is an antioxidant and thyroid function may be impacted and may affect vitamin C levels. In this review, we covered different diseases such as Graves' disease, goitre, thyroid cancer and other causes of hyperthyroidism and hypothyroidism.
Additionally, a study of vitamin C and other drugs like levothyroxine was conducted. Vitamin C may rectify anomalies in serum-free T4, T3

ACK N O WLE D G E M ENTS
None.

FU N D I N G I N FO R M ATI O N
None.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I LT Y S TAT E M E N T
Not applicable.