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Mechanisms of Disease: hydrogen peroxide, DNA damage and mutagenesis in the development of thyroid tumors Knut Krohn, Jacqueline Maier and Ralf Paschke* INTRODUCTION

S U M M A RY Somatic mutations can be identified in two-thirds of papillary and follicular thyroid carcinomas and ‘hot’ thyroid nodules, whereas equivalent mutations relevant for benign ‘cold’ thyroid nodules are unknown. This Review summarizes current knowledge about early molecular conditions for nodular and tumor transformation in the thyroid gland. We reconstruct a line of events that could explain the predominant neoplastic character (i.e. originating from a single mutated cell) of thyroid nodular lesions. This process might be triggered by the oxidative nature of thyroid hormone synthesis or additional oxidative stress caused by iodine deficiency or smoking. If the antioxidant defense is not effective, this oxidative stress can cause DNA damage followed by an increase in the spontaneous mutation rate, which is a platform for tumor genesis. The hallmark of thyroid physiology—H2O2 production during hormone synthesis—is therefore very likely to be the ultimate cause of frequent mutagenesis in the thyroid gland. DNA damage and mutagenesis could provide the basis for the frequent nodular transformation of endemic goiters. Keywords hydrogen peroxide, mutagenesis, oxidative stress, thyroid, tumor

Review criteria We searched PubMed for publications using different combinations of the search terms “thyroid”, “nodule”, “tumour”, “benign”, “clonality”, “clonal origin”, “mutagenesis”, “gene mutation”, “point mutation”, “genetic variation”, “mutation rate”, “DNA mutational analysis”, “DNA modification”, “DNA damage”, “DNA lesion”, “base damage”, “adducts”, “derivatives”, “deamination”, “DNA oxidation”, “oxidative attack”, “oxidative damage”, “oxidative stress”, “reactive oxygen species”, “free radicals”, “radicals”, “radical induced”, “H2O2”, “DNA repair”, “base excision repair”, “repair enzymes”, “DNA glycosylase”, “endonuclease”, “base pairing”, and “base pair mismatch”. All selected papers were English-language, full-text articles. A number of references were not included because of space restrictions.

K Krohn is Head of the DNA Technology Core Facility of the Interdisciplinary Center for Clinical Research, J Maier is a Doctoral Fellow at The Third Medical Department and R Paschke is Professor of Endocrinology at the Third Medical Department, University of Leipzig, Leipzig, Germany. Correspondence *Medical Department III, University of Leipzig, Philipp Rosenthal-Strasse 27, D-04103 Leipzig, Germany [email protected] Received 8 May 2007 Accepted 24 July 2007 www.nature.com/clinicalpractice doi:10.1038/ncpendmet0621

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Thyroid nodules are a frequent diagnosis in thyroid clinics. Although they are predominantly benign, assessing the risk of thyroid nodule malignancy is a major diagnostic challenge. The current medical treatment options for benign nodules have a limited evidence base. Treatment options and the discrimination between benign and malignant nodules are mainly hampered by the lack of understanding of the molecular etiology of benign nodules. Etiologically relevant somatic mutations can be identified in two-thirds of papillary and follicular thyroid carcinomas and hyperfunctional (‘hot’) thyroid nodules. By contrast, somatic mutations relevant for benign, hypofunctional (‘cold’) or normofunctional (‘warm’) thyroid nodules, which constitute the majority, are unknown. The purpose of this Review is, therefore, to report current knowledge concerning early molecular conditions for nodular and tumor transformation in the thyroid. We will outline a sequence of molecular events that include oxidative stress and DNA damage as the trigger for somatic mutations in the thyroid gland. Particular hallmarks of thyroid physiology, including thyroid hormone synthesis and H2O2 production, are very likely to be the ultimate cause for frequent mutagenesis in the thyroid gland. In fact, the oxidative burden seems to be a potential initial starting point for DNA damage and the formation of somatic mutations, especially in iodine-deficient regions. We will, therefore, also summarize the influence of nutritional factors such as iodine on oxidative stress, DNA damage and mutagenesis. THYROID NODULES

Studies that use sensitive ultrasound methods detect nodules in about 50% of thyroid glands.1,2 A similar proportion is detected in postmortem evaluations.3 As a general classification, growths occur as solitary nodules or are multinodular. Our more detailed working hypothesis concerning the classification of thyroid nodules is summarized in Figure 1. In terms of functional activity—as tested by radioactive iodine or technetium uptake on

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A Hyperfunctional Thyroid tumor Normofunctional Carcinomas

Normal thyroid glands

Hypofunctional Hyperplastic nodules

Single cell Focal intrinsic activation (e.g. somatic mutation)

True tumors (clonal origin) B

Normal thyroid glands

Figure 1 The classification of thyroid nodules on the basis of results from clonality studies. Such studies imply that the majority of thyroid nodules are ‘true’ thyroid tumors as opposed to polyclonal, hyperplastic nodules. Traditionally, only thyroid adenomas are considered true tumors, on the basis of an exclusive histologic definition—the presence of a capsule and a growth pattern that is different from the surrounding normal parenchyma in an otherwise normal thyroid gland. Strict histologic criteria are, however, difficult to obtain in the frequent presence of goiter or thyroiditis. The biological basis for separating hyperplastic thyroid nodules from true tumors should, therefore, also depend on their clonality.65 In the light of the many thyroid nodules without a capsule (adenomatous nodules) that are monoclonal, a mixed functional and molecular definition of true thyroid tumors seems more objective and consistent.

scintiscan—hyperfunctional, normofunctional or hypofunctional entities can be differentiated. There is consensus that the percentage of malignant neoplasias (carcinomas) among thyroid nodules is in the order of 5% for patients in thyroid clinics;4 moreover, papillary microcarcinomas are a frequent finding in autopsy studies.5 There are at least three lines of evidence suggesting that most nodular lesions are benign tumors that prevalently arise from a single progenitor cell that contains a defect (Figure 2). First of all, constitutively activating somatic mutations in the TSH receptor (TSHR) or the Gsα protein are the major cause of autonomously functioning thyroid nodules.6,7 Here, “autonomously” refers to the ability to function without TSH. Autonomously functioning thyroid nodules are usually hot; however, warm

Monoclonal lesion Polyclonal lesion

Focal extrinsic activation (e.g. growth factor stimulation)

Thyroid hyperplasia

Figure 2 The two concepts of thyroid nodular transformation. (A) A monoclonal lesion starts from a single cell with a genetic aberration whereas (B) a polyclonal lesion or hyperplasia develops from a group of cells under extrinsic stimulation.

thyroid nodules can also seem autonomous in suppression sintigraphy. Secondly, a frequently monoclonal origin is suggested from studies of thyroid adenomas or adenomatous nodules diagnosed as single lesions or within multinodular goiters in areas where goiters are endemic.8 Despite some technical concerns, for example identification of monoclonality in very small cell populations of fixed tissue sections using polymerase chain reactions (discussed by Krohn et al.8), a monoclonal origin often suggests a somatic genetic change (e.g. a point mutation or gene rearrangement) transferred from the founder cell of the clone. Thirdly, somatic mutations can already be present in microscopic areas comprising only a few thyroid follicles in sections of euthyroid goiters.9 Because of the slow proliferation rate of thyroid epithelial cells, a long period (tens of years) between initiation of the tumor and nodular

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appearance would seem to be very likely. In this regard, the frequency of thyroid tumors is a paradox that can only be explained by frequent tumor initiation and/or by enhanced thyroid epithelial cell proliferation. The origin of tumor formation in the thyroid gland could therefore be a natural or induced high mutation rate and aberrant growth stimulation. The latter very probably involves endogenous growth factors or exogenous goitrogenic substances. Notably, nodular transformation of enlarged thyroid glands is a common clinical finding. Proliferation is very important for the manifestation of mutagenesis, and DNA replication during cell division leads to a fixation of spontaneous mutations into the genome, causing a certain mutation load for dividing cells. Highly proliferating tissues—such as the colon, endometrium, skin, prostate, or breast—are, therefore, tumor-prone. Because the thyroid gland has relatively low proliferation, the tumor incidence should be much lower than is actually the case. THE Downside OF THYROID HORMONE SYNTHESIS

The main function of the thyroid gland is to synthesize the thyroid hormones l-3,5,3',5'tetraiodothyronine (T4) and l-3,5,3'-triiodothyronine (T3). To do so, the thyroid gland takes up iodine from food and incorporates it into thyroglobulin—the precursor of the thyroid hormones. Iodination of tyrosyl residues on thyroglobulin requires high concentrations of H2O2 and oxidized iodine, which are generated by the enzymes thyroid oxidase 1, thyroid oxidase 2 and thyroid peroxidase. Besides being a substrate in hormone synthesis, H2O2 could be a major source of free radicals and reactive oxygen species (ROS). Because these molecules can cause substantial damage to a cell and impair normal function, thyroid epithelial cells are likely to have a potent defense mechanism to counterbalance potential damage mediated by free radicals. It has been shown that antioxidant enzymes such as glutathione peroxidases (GPXs) or thyroid peroxidase are upregulated during thyroid hormone synthesis.10 GPX3 has, moreover, been suggested to directly interfere with thyroid hormone synthesis by affecting the concentration of H2O2.11 If antioxidant defense is not effective enough, excessive damage (e.g. peroxidation) should, therefore, be detectable in lipids, DNA and proteins of thyroid epithelial cells. october 2007 vol 3 no 10 KROHN ET AL.

DNA DAMAGE AND SPONTANEOUS MUTATION IN THE NORMAL THYROID

It has been estimated that the genome of a mammalian cell receives about 104–105 oxidative hits per day.12 H2O2 generated during thyroid hormone biosynthesis could add an extra hazard for the DNA of thyroid epithelial cells. Although the chemical compound H2O2 itself is almost inert towards DNA, it can give rise to highly reactive •OH radicals in a process that is catalyzed by transition metal ions, typically Fe2+, and is known as the Haber–Weiss and Fenton reaction.13 The •OH radical molecule is one of the ROS that is extremely reactive in the oxidation of cellular constituents. DNA oxidation that is induced by •OH causes five main classes of damage: oxidized bases,14 abasic sites, DNA– DNA intrastrand adducts,15,16 DNA strand breaks and DNA–protein cross-links.17,18 Because ROS have been of such importance for survival, cells have developed mechanisms to detect the presence of ROS accurately, in order to regulate metabolism and antioxidant responses.19 ROS-derived signals are used by cells to regulate growth or proliferation,20 differentiation,21,22 and death.19,23,24 In thyrocytes, H2O2-mediated cytotoxicity seems to be dose-dependent, requiring only low concentrations to result in thyroid cell apoptosis rather than necrosis (apoptosis is thought to protect from tumorigenesis).19,24 Findings in the thyroid glands of male Wistar rats suggest, furthermore, that the predominant cytotoxic response to oxidative stress might differ depending on the functional state of the gland.19,25 Although more than 20 modified DNA products and intermediates that are produced by ROS and free radicals have been identified (see Supplementary Table 1), 8-oxo-2'-deoxyguanosine (8-OHdG) is the most frequently investigated DNA adduct.26 Because of its mutagenic character and the high sensitivity of its immunological detection, 8-OHdG has become the best marker for oxidative DNA damage. In thyroid tissue sections, an antibody to 8-oxoguanine that also detects 8-OHdG and 8-oxoguanosine (the modified RNA nucleotide) shows the most prominent staining in the follicular cells near the lumen, where H2O2 is generated.27 Staining in the follicular cells of the thyroid seems, moreover, to be stronger than in spleen, lung and liver cells,27 which might indicate a higher load of oxidatively modified DNA, possibly caused by high H2O2 concentrations. nature clinical practice ENDOCRINOLOGY & METABOLISM 715

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A similar conclusion is supported by analysis of single-base DNA modifications detected with the comet assay. In principle, this assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the nucleus if subjected to an electric field, thereby forming a ‘tail’. Migration and hence the extent of the tail depends on the loss of DNA integrity (e.g. through strand breaks and abasic sites). In conjunction with the standard comet assay, optional in vitro treatment with repair enzymes causes formation of abasic sites and strand breaks at sites with specific DNA modifications.28 For the repair-enzyme protocols that detect oxidative attack on DNA, thyroid tissue shows the highest scores with the most prominent differences compared with liver, lung and spleen.27 In addition, staining for 8-OHdG and its modified RNA nucleotide using the antibody method described above perfectly agrees with the results of the modified comet assay that includes detection of 8-OHdG.27 Our hypothesis of excessive oxidative DNA damage in the thyroid gland is further supported by data on mRNA expression of the 8-oxoguanine DNA glycosylase gene (OGG1), which encodes the enzyme that would repair 8-oxoguanine modifications.29 As expected, OGG1 is highly expressed in lung—an organ with pronounced oxygen exposure. A very similar level of mRNA expression of OGG1 is detected in the thyroid, however—much higher than in spleen or liver.27 In addition, preliminary data suggest a reduced expression of OGG1 in follicular thyroid cancer and higher expression in Graves’ disease (S Karger et al., personal communication). Such a distribution would again correlate with the functional activity of thyroid cells and could indicate a higher load of ROS during hormone synthesis. Because many of the DNA modifications lead to non-Watson–Crick pairing of the affected bases, they are potentially mutagenic. For example 8-OHdG pairs preferentially with A rather than C, which can generate a G>T transversion;30 this transversion is predominantly found in genes associated with tumor development.31–33 Many lesions in DNA that are caused by oxidation are mutagenic, no matter whether they are formed in situ or misincorporated from the deoxynucleotide pool during replication. It is therefore very likely that the thyroid gland might constitute a mutagenic environment. Such a hypothesis has been tested in studies of the TSHR.34 Although the source of oxidative stress in thyroid carcinomas, cold benign thyroid 716 nature clinical practice ENDOCRINOLOGY & METABOLISM © 2007 Nature Publishing Group

tumors and autonomously functioning thyroid nodules might be different (e.g. hypoxia versus increased thyroid hormone metabolism), markers of oxidative stress are detectable in cold nodules.35 Hypermutability in human thyroid carcinomas compared with tumors in general36—and as demonstrated in studies of silent p53 mutations—could therefore be, at least in part, the result of oxidative DNA damage. We followed this line of reasoning by analyzing the spectrum of base exchanges in somatic mutations of hyperfunctional thyroid tumors that cause constitutive activation of the TSHR.27 Among the generally high frequency of transitions previously noted in mutation hot-spots,34 the predominance of C>T transitions in somatic mutations and higher frequencies of G>T and T>C base exchanges (both compared with the spectrum of germline TSHR mutations) further suggests a mechanism that is very probably caused by oxidized base adducts.37,38 If such an oxidative fingerprint is detectable in databases of TSHR and p53 mutations, then what about the spontaneous mutation rate (SMR) in the thyroid at large? To answer this question we determined the SMR using mice transgenic for a lacZ reporter construct. The lacZ transgene can be rescued from the genomic DNA of these mice and tested for spontaneous mutations that affect the enzymatic activity of the lacZ gene product (i.e. β-galactosidase).39 Most interestingly, we found a strikingly high SMR in the thyroid gland:27 with an 8–10-fold higher rate compared with liver, the thyroid stands out from many other tissues.40 Indeed, the SMR in the mouse thyroid glands without any experimental mutagenic challenge shows values that are usually only found in other organs (e.g. liver) in animals treated with mutagens like ethyl nitrosourea or benzo[a]pyrene.41 In summary, these data reveal a connection between thyroid hormone meta bolism, oxidative DNA damage and the SMR in the normal thyroid gland that could represent the basis for the frequent tumorigenesis. On top of this generally high mutation rate in the normal thyroid gland, environmental and lifestyle factors could add to the pool of DNA damage and increase mutagenesis and tumor initiation. Radiation-induced DNA-strand breaks leading to genetic rearrangements (e.g. between the proto-oncogene RET and the gene for coiled-coil domain containing 6 [CCDC6; previously known as PTC]) are a frequent molecular cause of papillary thyroid carcinoma KROHN ET AL. october 2007 vol 3 no 10


in regions affected by the Chernobyl nuclear accident.42 By contrast, point mutations in the BRAF (v-raf murine sarcoma viral oncogene homolog B1) gene, which could be the result of the high mutation rate in the normal thyroid gland, combined with increased proliferation, are the most common genetic alterations in patients without a history of exposure to radioactive iodine. An excellent overview concerning thyroid carcinomas that includes known genetic alterations was recently given by Kondo et al.43 Tobacco smoking has been reported as another risk factor for thyroid tumorigenesis.44 The proposed mode of action involves thiocyanateion-induced blockage of the iodine transport into the thyrocyte, which very probably leads to an intracellular iodine deficiency. How iodine deficiency could exacerbate oxidative stress in thyrocytes and increase mutagenesis will be the focus of the next section.

A Oxidative stress

Selenium

DNA damage

Antioxidant defence

ThOX 1 or 2 ROS

H2O2

Normal thyroid

Hormone synthesis TPO

T3, T4

Iodide

Mutagenesis B

?? Oxidative stress

Selenium deficiency

DNA damage

Antioxidant defence

ThOX 1 or 2 ROS

H2O2

Iodine and/or selenium deficiency

Hormone synthesis TPO

T3, T4

Iodide Mutagenesis ? Iodine deficiency

OXIDATIVE STRESS AND DNA DAMAGE IF THE THYROID LACKS IODINE

In addition to H2O2, iodine is another major substrate for the synthesis of the thyroid hormones that needs to be considered for its potential effects on thyroid tumor genesis. Iodide is found in soil and seawater and is oxidized by sunlight to iodine, which is vaporized into the air. Some of this iodine is lost in the stratosphere. These events account for the continued depletion of iodine in the soil and the lack of capture by plants. This process can cause iodine deficiency in humans, particularly at higher altitudes and in countries where salt is not supplemented with iodide. Iodine deficiency currently represents a significant public health problem for about 30% of the world’s population, in 110 countries. The consequences of iodine deficiency even in developed areas such as Europe are enormous.45 The iodine status worldwide is recapitulated by the WHO Global Database on Iodine Deficiency (see Delange et al.46). One of the most obvious clinical manifestations in geographical areas with iodine deficiency is thyroid enlargement—known as endemic goiter. This enlargement is caused by increased cellular proliferation that could provide a favorable milieu for tumor growth. It is not surprising that, in the long run, thyroid enlargement leads to nodular transformation of the thyroid and that nodular thyroid disease is more frequent in areas with iodine deficiency.47 Thyroid diseases that are related to iodine deficiency are not, however, restricted to nodules. They typically

Figure 3 Mechanisms that might cause mutagenesis in the thyroid gland. The figure shows the key molecules involved in those parts of thyroid hormone synthesis which—in conditions of iodine and most probably also selenium deficiency—lead to oxidative stress, DNA damage and possibly mutagenesis. (A) In the normal thyroid gland, the enzymes ThOX1and ThOX2 generate H2O2. TPO transfers oxidized iodine to tyrosyl residues of thyroglobulin—the precursor for T3 and T4 synthesis. H2O2 is, however, a source of ROS, which— with other oxidative stress—can cause DNA damage. Normally, antioxidant defense can prevent oxidative stress and DNA damage. Selenoproteins such as glutathione peroxidase 3 are part of this defense. (B) Conditions of iodine deficiency increase levels of H2O2 and might increase the amount of oxidative stress and DNA damage. Additional selenium deficiency decreases levels of selenoproteins and could thereby weaken antioxidative defense, and this exacerbates oxidative stress and DNA damage. Abbreviations: ROS, reactive oxygen species; ThOX, thyroid oxidase; TPO, thyroid peroxidase.

range from goiter to mild hypothyroidism and to the severest, but rare, form of cretinism. As outlined above, H2O2-mediated ROS generation is very likely to be an important starting point for thyroid tumor development (Figure 3). Because iodine and H2O2 act as co-substrates in thyroid hormone synthesis, changes of iodine concentration are very likely to affect the H2O2 concentration. In fact, generation of H2O2 is inhibited by iodide in vivo and in vitro.48,49 H2O2 generation—which is mandatory for the organification of iodine—is, moreover, stimulated by TSH, which increases the expression of genes important for thyroid hormone synthesis (e.g. genes encoding the sodium–iodine symporter or thyroid peroxidase).50 Low iodine levels and markers of increased thyroid functionality

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Iodine deficiency Selenium deficiency

Goitrogens

Tobacco smoking

Genetic susceptibility Age

Pregnancy

Goiter

H 2O 2

Hyperplasia

Radicals

Thyroid enlargement

DNA damage

Proliferation

Mutagenesis

Thyroid hormone metabolism

Figure 4 Factors that might contribute to the development of thyroid tumors. Thyroid cell proliferation and mutagenesis are the two conditions that are most important for the development of thyroid tumors. Environmental, lifestyle, genetic and metabolic factors can lead to proliferation and/or mutagenesis. Pregnancy and goitrogens can cause increased thyroid cell proliferation, leading to hyperplasia, which manifests as goiter or thyroid enlargement. Tobacco and normal hormone metabolism generate H2O2 and oxygen radicals, which—if uncontrolled—can cause DNA damage and mutagenesis. Iodine deficiency, selenium deficiency, genetic susceptibility and increasing age can cause both increased cell proliferation and mutagenesis.

suggest activation of H2O2 generation, which could result in DNA damage and somatic mutation.51 Low iodine and high H2O2 levels should, therefore, activate antioxidative defense, and this activation should be detectable in the cellular regulation of enzymes involved in defense against oxidative stress. Enzymes involved in H2O2 and superoxide detoxification should represent prominent markers for such a scenario. Indeed, a higher expression of mRNA for superoxide dismutase 3 (SOD3)—the extracellular SOD isoform that preferentially acts in the lumen, where H2O2 is generated—is detected during experimental iodine deficiency in mice.52 In addition, increased total SOD enzyme activity in thyroid glands exposed to lower iodine concentrations is evident;52 moreover, expression of mRNA for the glutathione-dependent enzymes (i.e. GPX3, GPX4 and glutathione S-transferase) and two peroxiredoxins (i.e. peroxiredoxin 3 and peroxiredoxin 5) changes during iodine deficiency.52 In particular, peroxiredoxins have been connected to H2O2 detoxification,53 and peroxiredoxin 5 shows a strongly increased 718 nature clinical practice ENDOCRINOLOGY & METABOLISM © 2007 Nature Publishing Group

immunolabeling in goitrous rats fed a lowiodine diet. Interestingly, upregulation of peroxi redoxin 2 and peroxiredoxin 6 has been shown in cold thyroid nodules35 that were associated with intranodular iodine deficiency.54 Differential expression of antioxidant enzymes underlines the importance of the antioxidant defense in the iodine-deficient thyroid gland; moreover, glutathione peroxidases are seleniumcontaining proteins. It is therefore very likely that selenium deficiency could impair the antioxidant defense and exacerbate oxidative stress (Figure 3). In turn, higher concentrations of ROS could cause DNA damage. The comet assay revealed a significant increase of uracil and oxidized purine or pyrimidine adducts in thyroid DNA during iodine deficiency.55 The increase of uracil modifications under iodine restriction could be the basis for the high frequency of C>T base changes in TSHR mutations that are found in autonomously functioning thyroid nodules in iodine-deficient areas.56 Uracil in DNA might arise from spontaneous hydrolytic or enzymatic deamination of cytosine or from misincorporation of deoxyuracil triphosphate instead of deoxythymine triphosphate during DNA replication.57–59 Whereas deamination of cytosine generates G:U mismatches that cause G:C to A:T transitions,60–62 misincorporation of deoxyuracil monophosphate during DNA replication would not cause a base change. Our theory of an increased oxidative burden in the thyroid gland through iodine deficiency is also supported by results of the comet assay with repair-enzyme protocols to detect oxidative DNA damage.55 For example, one of these modifications (i.e. 5-formyl-2'deoxyuridine) induces T>C and C>T transitions as well as G>T transversions,63 which are also very prominent in the spectrum of somatic, activating TSHR mutations found in autonomously functioning thyroid nodules.27 C>T transitions are, furthermore, the result of another set of cytosine modifications (i.e. 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol) detected with the modified assay.28,64 In normal thyroid DNA, we showed a correlation of DNA damage and the SMR.27 Although the SMR is very high in the normal thyroid gland, it is not changed in experimental iodine deficiency. This finding could either indicate a weakness of the experimental model, which has been discussed in detail elsewhere,27 or might KROHN ET AL. october 2007 vol 3 no 10


indicate a more effective repair of DNA damage in response to iodine deficiency. So far no data concerning DNA repair are available for the thyroid gland. CONCLUSIONS

In this Review, we have summarized current knowledge concerning early molecular conditions for nodular and tumor transformation in the thyroid gland (Figure 4). We have outlined a sequence of molecular events that include oxidative stress and DNA damage as the trigger for somatic mutations. The oxidative burden is already detectable in the normal thyroid gland and is very likely to be linked to hormone synthesis and H2O2 production. Environmental conditions (e.g. iodine deficiency) have the potential to make this situation deteriorate, however. In general any external factor (e.g. smoking) that would increase oxidative stress, cause DNA damage or increase the SMR might aggravate the chances for tumor genesis. On the other hand, any factor that increases proliferation (e.g. goitrogens) very probably shortens the time to develop a detectable tumor or nodule. Supplementary information, in the form of a table listing the known oxidative DNA-base modifications and their consequences for mutations, is available on the Nature Clinical Practice Endocrinology & Metabolism website. KEY POINTS ■

Histologically, thyroid nodules can be hyperplastic nodules or true tumors; both can be classified as hypofunctional, normofunctional or hyperfunctional, and the latter include carcinomas

■

The high frequency of thyroid tumors despite a slow proliferation rate of thyroid epithelial cells suggests a high mutation rate and/or a susceptibility to aberrant growth stimulation

■

H2O2 is crucial for the generation of thyroid hormones but is a potential source of reactive oxygen intermediates, which can lead to DNA damage if antioxidant defenses are inadequate

■

Iodine deficiency, selenium deficiency or tobacco smoking can increase levels of H2O2 and decrease antioxidant defenses

■

Conditions such as iodine deficiency, pregnancy and goitrogens can increase thyroid cell proliferation and contribute to the high rates of thyroid tumors

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