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Endocrinology Vol. 147, No. 7 3391-3397
Copyright © 2006 by The Endocrine Society

Deoxyribonucleic Acid Damage and Spontaneous Mutagenesis in the Thyroid Gland of Rats and Mice

J. Maier, H. van Steeg, C. van Oostrom, S. Karger, R. Paschke and K. Krohn

III Medical Department (J.M., S.K., R.P., K.K.) and Interdisciplinary Centre for Clinical Research (J.M., K.K.), University of Leipzig, D-04103 Leipzig, Germany; and Laboratory of Toxicology, Pathology, and Genetics (H.v.S., C.v.O.), National Institute of Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands

Address all correspondence and requests for reprints to: Knut Krohn, Ph.D., Interdisziplinäres Zentrum für Klinische Forschung Leipzig, University of Leipzig, Inselstrasse 22, D-04103 Leipzig, Germany. E-mail: krok{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid tumors are a frequent finding not only in iodine-deficient regions. They are predominantly characterized by somatic genetic changes (e.g. point mutations or rearrangements). Because slow thyroid proliferation is a apparent contradiction to a high frequency of tumor initiation, we characterized mutational events in thyroid. First we studied the frequency of certain base exchanges in somatic TSH receptor (TSHR) mutations and determined the spontaneous mutation rate in thyroid and liver. Then we applied different protocols of the comet assay to quantify genomic DNA damage and conducted immunohistochemistry for 8-oxoguanine as a molecular marker for oxidative stress. Among 184 somatic mutations of the human TSHR found in thyroid tumors, C->T transitions had a unexpectedly high frequency (>32%). The mutation rate in thyroid is 8–10 times higher than in other organs. The comet assay detected increased levels of oxidized pyrimidine (2- to 3-fold) and purine (2- to 4-fold) in thyroid, compared with liver and lung, and a 1.6-fold increase of oxidized purine, compared with spleen. Immunohistochemistry revealed high levels of 8-oxoguanine in thyroid epithelial cells. We have shown a strikingly high mutation rate in the thyroid. Furthermore, results of the comet assay as well as immunohistochemistry suggest that oxidative DNA modifications are a likely cause of the higher mutation rate. It is possible that free radicals resulting from reactive oxygen species in the thyroid generate mutations more frequently. This is also supported by the spectrum of somatic mutations in the TSHR because more frequent base changes could stem from oxidized base adducts that we detected in the comet assay and with immunohistochemistry.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID NODULES ARE very common. They occur in more that 50% of the normal population (reviewed in Ref. 1). There is consensus that the percentage of malignant neoplasias in these nodules can be up to 5% (2). However, the rates of benign neoplasm could be higher but are still controversial. Studies of thyroid adenomas or adenomatous nodules diagnosed as single lesion or within multinodular goiter in endemic areas suggest a frequent monoclonal origin (reviewed in Ref. 3) for hyperfunctional and hypofunctional entities. Another line of evidence that suggests a high frequency of tumor initiation in thyroid cells comes from microlesions. Microcarcinomas have been detected with a high frequency (4). They have recently been shown to be predominantly monoclonal and to derive from individual progenitor cells (5). Finally, we found somatic mutations in microscopic areas comprising only a few thyroid follicles in sections of euthyroid goiters that do not harbor tumors (6).

In contrast to a high frequency of neoplasms thyroid tissue is only slowly proliferating. In dog and human adult thyroid, the mitotic index for cells in the S phase are 39.5 x 10–5 and 13.4 x 10–5, respectively (7). This translates into an estimated time between cell divisions of thyrocytes of about 8.5 yr in humans and 2.3 yr in dogs (7), meaning that thyroid cells divide about five times during adulthood. Although mitotic rates in rat thyroid are higher than in dog and human, the number of thyrocyte divisions during adulthood is slightly lower (8). In the context of mutagenesis, proliferation is very important. DNA replication during cell division leads to a fixation of spontaneous mutations into the genome, causing a certain mutation load for dividing cells. Hence, compared with highly proliferating and therefore tumor-prone tissues such as the colon, endometrium, skin, prostate, or breast at a similar mutation rate, the tumor incidence in the thyroid should be much lower than actually occurring. To find a possible explanation for this difference, we studied thyroid spontaneous mutagenesis. Strikingly, we show in this study that the spontaneous mutation rate (SMR) in the thyroid is much higher than in other tissues. This could be due to the burden of thyroid hormone synthesis put onto the thyroid cell, which involves generation of free radicals and reactive oxygen species. Excessive oxidative stress might cause DNA damage and somatic mutations. To elucidate such a possible molecular cause for a higher SMR, we studied 8-hydroxydeoxyguanosine/8-hydroxyguanosine (8-OHdG/8-OHG) concentration in thyroid DNA as a marker of DNA modification. We also used different modifications of the comet assay to characterize DNA damage in the thyroid gland as a possible cause of extensive mutagenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
To study the rate of in vivo mutations, we used transgenic mice (mixed-sex C57BL/6) containing a lacZ reporter plasmid [pUR288 (9)]. In addition, BALBC mice and Wistar rats were used to determine DNA modifications. For all experiments adult mixed-sex animals were fed a regular diet (purified C1000; Altromin, Lage, Germany). Water and diet were supplied ad libitum. Ethical approval was given by the regional board.

Tissue preparation
Animals were killed by a carbon dioxide atmosphere at an age of about 2–3 months (C57BL/6 also at 1 yr). Serum and thyroid tissue as well as samples from other organs (e.g. liver, lung, spleen) were collected, immediately frozen on dry ice, and stored at –80 C or kept at 4 C until further processing. For the comet assay, the fresh tissue was minced in mincing solution (pH 7.5) [Hanks’ balanced salt solution (Ca2+, Mg2+ free), 20 mM EDTA, and 10% dimethylsulfoxide (DMSO)] and aliquots were frozen in liquid nitrogen.

DNA and RNA preparation
Genomic DNA was isolated using the DNA isolation kit from Roche Diagnostics (Mannheim, Germany) based on cell lysis, proteinase K digest, DNA binding to silica surface and magnetic bead separation according to the protocol of the manufacturer.

Total RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Afterward, the total RNA was purified with RNeasy columns (QIAGEN, Hilden, Germany) according to the RNA clean-up protocol.

SMR
To rescue the plasmid-derived DNA sequences (pUR288) containing a lacZ reporter from genomic DNA, we followed the protocol by Gossen et al. (10). Briefly, genomic DNA from different organs of C57BL/6 transgenic mice was digested with the restriction enzyme HindIII in the buffer suggested by the manufacturer (MBI Fermentas, Roth, Germany). Afterward lacI repressor bound to magnetic beads was used to separate the plasmid from genomic DNA. After two washing steps, plasmid DNA was eluted with isopropylthio-ß-galactosidase. Linear pUR288 DNA was circularized with T4 ligase for 2 h, precipitated, and electroporated into electrocompetent Escherichia coli C cells ({Delta}lacZ/galE) using a gene pulser in combination with a pulse controller (Bio-Rad, Richmond, CA) at a setting of 2.5 kV, 25 µF, and 200 {Omega}. Aliquots of E. coli cells were plated on LB agar plates to determine the titer and on LB agar plates with the lactose-analog P-gal, which allows colony formation only in lacZ– cells and hence mutation detection (11).

8-OHdG/8-OHG immunohistochemistry (IHC)
Four- to 7-µm slices of different organs were cryo-cut with a Cryostat (Leica, Nussloch, Germany), mounted on slides (SuperFrost Plus Gold; Menzel, Braunschweig, Germany), dried 20 min at room temperature, and kept until further processing at –20 C. To allow a better comparison, sections of all tissues were mounted on one slide for later processing.

Before IHC the slides were incubated for 5 min at 68 C, fixed for 90 sec with chilled acetone, and rinsed in Tris-buffered saline (TBS) buffer. After blocking with Dako protein block (code X0909; Dako, Carpinteria, CA) for 30 min, sections were incubated for 1 h with a primary polyclonal goat anti-8-OHdG/8-OHG antibody (AB5830; Chemicon International, Temecula, CA). The primary antibody was diluted 1:200 in DakoCytomation antibody diluent (Dako). A DakoCytomation LSAB+ system-AP kit was used for detection. Sections were washed three times in TBS and linked for 30 min with biotinylated link antibody. After thorough washing in TBS, an alkaline phosphatase-labeled streptavidin was applied for 30 min at room temperature. Sections were rinsed in TBS and incubated with fuchsine-chromogene solution as phosphatase substrate for 10 min at room temperature. Because 8-OHdG is localized in the nucleus and could therefore interfere with counterstaining, only a subset of sections were counterstained with hematoxylin. For a negative control, sections were incubated without the primary antibody. H2O2-treated sections were used a positive control. Moreover, thyroid tissue from Graves’ disease patients with hyperactive hormone synthesis showed increased staining for 8-OHdG/8-OHG (Karger, S., and D. Führer, unpublished data).

Semiquantitative evaluation of 8-OHdG and 8-OHG
Identically processed sections of the four tissues from one animal per slide were compared for overall staining intensity by three experienced researchers using a scale from 1 to 4. Because blinding was not possible due to the histological characteristics of the four tissues, the scoring was strictly based on the evaluation of individual cells. Sections from four animals were evaluated, and the average calculated for each tissue was expressed as staining index. The results of the different researchers were highly reproducible for all animals studied.

Comet assay
We followed the protocol described by Miyamae et al. (12) and Tice et al. (13) with some modifications for the detection of uracil and oxidized bases using repair enzymes (14). To prevent additional DNA damage, we protected the tissue from UV and strictly kept all solutions on ice. Small pieces (5 mg) of an organ were washed and minced into very small pieces in mincing solution (pH 7.5) [Hanks’ balanced salt solution (Ca2+, Mg2+ free), 20 mM EDTA, and 10% DMSO]. Three microliters of this suspension were gently mixed with 85 µl of 0.6% low melting point agarose in PBS and placed on a SuperFrost microscope slide (Roth, Karlsruhe, Germany) coated with 1% normal agarose. After adding a coverslip, the agarose was permitted to solidify in a refrigerator for 10 min. Afterward 0.6% normal agarose/PBS was layered on top of the cells. Solidified slides were immersed in chilled lysing solution (pH 10) (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% sodium N-laurylsarcosinate, 10% DMSO, 1% Triton X-100) at 4 C for 60 min. After lysis the slides were washed three times in enzyme reaction buffer at 4 C. Before restriction excess liquid was removed. Fifty microliters of enzyme solution (endonuclease III, formamidopyrimidine DNA glycosylase (FPG), and uracil-DNA-glycosylase (UDG); Sigma-Aldrich; 0.02 U/µl) or buffer alone, as control, was placed onto the gel and covered with a coverslip. Controls and enzyme-treated samples were incubated in a moist box at 25 C for 10 min following at 30 C for 30 min (endonuclease III, FPG) and 37 C for 60 min (UDG). Then the slides were placed on a horizontal gel electrophoresis platform on ice and covered with chilled alkaline solution (300 mM NaOH, 1 mM Na3 EDTA) for 20 min to unwind the DNA and break open alkali labile sites. After electrophoresis [17 min at 26 V/300 mA (1 V/cm)], the slides were neutralized for 3 x 5 min in neutralization solution [400 mM Tris-HCL (pH 7.5)] and then dried in air or directly stained with SYBR Green (Sigma-Aldrich) for 20 min.

To classify the comets into different types (12), we used a fluorescence microscope at a magnification of x200 and examined a minimum of 100 comets per organ per animal. Fife types were defined according to Fig. 1Go. All determinations were repeated at least once. The comet score was calculated in modification (to Ref. 15) as sum of the percentage of each type n (running from 1 to 5) times n-1. In our examination the score reached values between 0 and 400. Scores for the modified comet assay procedures are expressed as the differences to the comet assay procedure alone.


Figure 1
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  		FIG. 1. Classification of comets by shape: type 1, no tail; type 2, tail length less than a quarter of the head diameter; type 3, tail length between a quarter and a full head diameter; type 4, tail length greater than head diameter; type 5, comets with poorly defined or small head.

 
Real-time RT-PCR
The quantification of mRNA expression of DNA repair genes by real-time RT-PCR was performed using a LightCycler (Roche, Mannheim, Germany) as previously described (16). The nucleotide sequences of the primers and PCR conditions are available on request. The mRNA expression of the investigated genes was calculated as the mean ± SEM (n = 4) of the number of transcripts for the respective tissue divided by the mean number of transcripts in all four tissues after normalization to S6 ribosomal protein.

Statistics
Differences of SMR and comet scores were analyzed with one way ANOVA. Student’s t test was used for pair-wise comparison of SMR values. Tukey’s multiple comparison test embedded in the GraphPad Prism 4.03 software package (GraphPad Software Inc., San Diego, CA) was used for statistical analysis of comet scores of different tissues. The statistical error is expressed as SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of somatic TSH receptor (TSHR) mutations
The TSHR Mutation Database II (http://www.uni-leipzig.de/~innere/tsh/) lists 184 cases of hyperfunctioning thyroid adenoma, adenomatous nodules, or carcinomas with a somatic mutations of the human TSHR (17). The frequency of a certain base exchange is summarized in Fig. 2Go. Especially C->T transitions have an unexpectedly high frequency (>32%) about 4 times the statistical average of 8%, which would indicate equal frequency of all possible exchanges. C->T exchanges predominantly occur in codons A623 and T632 in which the exchanges GCC->GTC and ACC->ATC have been independently reported in seven and 17 cases, respectively. Moreover, G->T transversions (14%) and T->C transitions (12%) are also very distinct from all remaining exchange variants.


Figure 2
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  		FIG. 2. Frequency of base exchanges in human somatic TSHR mutations from 184 cases of hyperfunctioning thyroid adenoma, adenomatous nodules, or carcinomas listed in the TSHR Mutation Database II (http://www.uni-leipzig.de/~innere/tsh/).

 
SMR in the thyroid
Mice transgenic for a lacZ reporter construct based on the plasmid pUR288 were used to determine the SMR in the thyroid. Figure 3Go shows the number of mutant transgene lacZ copies vs. wild-type copies for genomic DNA from thyroid gland and liver for comparison. Numbers for the SMR in the thyroid gland range from 56 x 10–5 to 72 x 10–5 mutants per wild-type sequence. We did not detect a significant difference between young adult and older mice. In contrast, the mutation rate in liver is significantly lower (P < 0.01) with a number of 6.7 x 10–5 (9.6% of the thyroid level).


Figure 3
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  		FIG. 3. SMR was studied in the thyroid gland of transgene lacZ reporter mice at the age of 3 months to 1 yr (black bars). For comparison, SMR in liver (white bar) was determined after 3 months. Given are the average numbers and SEM (n = 6–7) of mutated lacZ molecules per 105 wild-type lacZ molecules. Differences of thyroid tissues vs. liver are highly significant (P ≤ 0.01).

 
Comet assay
This single-cell assay was used to assess the DNA damage at the steady-state level in thyroid, liver, lung, and spleen of 12- to 16-wk-old rats. Comet scores with and without prior enzymatic digestion are given in Fig. 4Go. Without further digest the comet assay allows to quantify DNA strand breaks and alkaline labile sites such as apurinic/apyrimidinic sites. A comet score of 60 ± 8 for thyroid is at the lower end, whereas spleen (104 ± 13) and lung (91 ± 9) are very similar. Only tissue from liver shows a significantly increased score (145 ± 16, P < 0.001).


Figure 4
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  		FIG. 4. Steady-state level of DNA damage detected with the comet assay (A) or different modifications of this assay (B and C). Cell nuclei were prepared from the thyroid gland (black bars), liver (white bars), lung (diagonal striped bars), and spleen (vertical striped bars) of 3-month-old Wistar rats. The comet (A) assay detects DNA strand breaks and alkaline labile sites. UDG modification (B) detects uracil bases in nuclear DNA. Modification with endonuclease III (E III) (C) and FPG (D) detects oxidation-related base adducts. All data are expressed as mean ± SEM (n = 4). Significant differences were assessed by ANOVA with repeated measures followed by Tukey’s multiple comparison test. Stars represent the level of significance, compared with thyroid. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

 
By combining the comet assay with a digest by DNA repair enzymes, it is possible to detect certain classes of DNA modification. Here we used the enzymes UDG, endonuclease III, and FPG. Data for these modifications are given as comet score with enzyme digest minus score of comet assay alone.

UDG catalyzes the cleavage of uracil base residues from both single- and double-stranded DNA and leaving the DNA phosphodiester backbone intact (18, 19). The resulting abasic site will cause a strand break in the following steps of the comet assay procedure (denaturation and electrophoresis). The comet scores for the DNA uracil content (UDG modification) range from 29 ± 14 for liver to 171 ± 29 for spleen. Thyroid and lung are in the middle of this range (Fig. 4Go).

Endonuclease III is an iron-sulfur-containing DNA repair enzyme (20) that removes pyrimidine modifications like thymine glycol, 5-hydroxycytosine, and 5,6-dihydrothymine (21, 22). It also lyses abasic sites (23). Endonuclease III-modified comet assay detects a 2.4-fold higher and 1.9-fold higher content of pyrimidine adducts in the thyroid with a comet score of 140 ± 15, compared with liver (comet score 59 ± 10, P < 0.001) and lung (comet score 73 ± 7, P < 0.01), respectively. The comet score of 127 ± 16 for spleen is not significantly different from thyroid.

FPG protein is a key enzyme in the DNA base excision repair pathway (24). It catalyzes the excision of a broad spectrum of modified purines (e.g. formamidopyrimidine and 8-OHdG). FPG-modified comet assay detects a 2.2-, 3.9-, and 1.6-fold higher content of modified purines, compared with liver (comet score 78 ± 12, P < 0.001), lung (comet score 44 ± 9, P < 0.001), and spleen (comet score 106 ± 14), respectively.

In control experiments we included oxidative challenge by H2O2 and use of antioxidants in the cell preparation protocols to test the effect of the preparation procedure on DNA integrity. Addition of H2O2 to the preparation had a clear effect, resulting in higher comet scores through strand breaks and/or abasic sites. In contrast, addition of antioxidants to the preparation buffer before the preparation did not change the comet scores in preparations without any exogenous H2O2 spike.

IHC for 8-OHdG/8-OHG
The antibody we used in our study recognizes 8-oxoguanine in both 8-OHdG and 8-OHG. It therefore labels modified DNA and RNA. In the thyroids of Wistar rats, labeling is most intensive in epithelial cells around the lumen (Fig. 5Go). Staining of the thyroid parenchyma is weaker. On the cellular level, stronger staining is detected in the cytosol, which implies oxidative modification of RNA or DNA of mitochondria.


Figure 5
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  		FIG. 5. Thyroid, liver, lung, and spleen of 3-month-old Wistar rats were analyzed by IHC (red color) using an antibody against 8-oxoguanin present in 8-OHdG/8-OHG as described in Materials and Methods. A, Negative control. B, Counterstained with hematoxylin. C, Without counterstaining. D, Mean staining intensity of 8-OHdG/8-OHG was evaluated as described in Materials and Methods on a scale from 1 to 4. Data are expressed as mean ± SEM (n = 4) of three independent evaluations. Intense staining is detected in thyroid follicular cells (black arrowheads).

 
In contrast to thyroid, staining in the liver is more homogeneous. Capillaries and bile ducts show more intense staining. Spleen is more difficult to evaluate because the cell density is higher, compared with the other tissues. We refrained from quantifying red pulpa because it shows prominent red staining already in the negative control.

The staining index calculated from visual examination by three experienced researchers (for details see Materials and Methods) is given at the bottom of Fig. 5Go. Thyroid reaches the highest index value, compared with the other organs.

mRNA expression of DNA-(apurinic or apyrimidinic site) lyase (APEX1) and 8-oxoguanine DNA glycosylase (OGG)-1
Complementary to the comet assay and IHC for 8-OHdG/8-OHG, we studied the mRNA expression of two DNA repair genes (APEX1 and OGG-1) in thyroid, liver, lung, and spleen (Table 1Go). After normalization to the expression of S6 in the respective tissue, there are differences of the expression of APEX1 and OGG1 in the four tissues. APEX1 expression is higher in thyroid and liver, compared with lung and spleen, whereas OGG1 expression is slightly higher in lung and thyroid, compared with liver and spleen.


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  		TABLE 1. mRNA expression of DNA repair genes

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to characterize molecular events that would explain a high frequency of somatic mutations and tumor initiation in the thyroid gland. Most interestingly, we found a strikingly high SMR in the thyroid. With an 8- to 10-fold higher number, compared with liver, the thyroid stands out from many other tissues (25). Indeed, SMR in the mice thyroids without any experimental mutagenic challenge shows values that are usually found only in animals treated with mutagens like ethyl nitrosourea (9) or benzo[a]pyrene (26). One possible cause for a higher SMR could be thyroid hormone synthesis itself because it produces H2O2 for the organification of iodine. H2O2 could be a major source of free radicals and reactive oxygen species resulting in DNA damage and somatic mutation. The hypothesis that the thyroid might comprise a mutagenic environment has been suggested in studies of TSHR (27) and p53 mutations (28, 29). Strikingly, rates of silent mutations in p53 suggest hypermutability in human thyroid tumors, compared with tumors in general (29). We followed this line of thinking by analyzing the spectrum of base exchanges in somatic mutations of thyroid tumors causing constitutive activation of the TSHR. Among the general high frequency of transitions previously noted in mutation hot spots (27), the predominance of C->T transitions in somatic mutations, compared with the spectrum of germline TSHR mutations and higher frequencies of G->T and T->C base exchanges, suggests a mechanism that is very likely caused by oxidized base adducts (30, 31, 32). Although C->T transitions are the most common point mutations in many genes, none of the C->T transitions found in the TSHR occur at CpG dinucleotides. This excludes the frequent deamination of 5-methylcytosine to thymidine, which is a prevalent mechanism of mutagenesis. Because the TSHR has a low prevalence of CpG in its mutable regions (27), we compared our finding with the spectrum of p53 point mutations in thyroid cancers. With a frequency of 29%, non-CpG C->T transitions are much more frequent in thyroid tumors than in all other tumors (19%) found in p53 databases (29).

To further substantiate our hypothesis of oxidative mechanisms as the cause of mutagenesis in the thyroid, we studied the extent of DNA damage. We applied different protocols of the comet assay to detect certain forms of DNA modifications that would allow to conclude a causative mechanism. In principle, the assay is based on 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). Optional in vitro treatment with repair enzymes causes formation of abasic sites and strand breaks at sites with specific DNA modifications. Concerning such modifications, differences between thyroid and liver, lung and spleen are most prominent for the endonuclease III and FPG protocols, which implies oxidative attack on DNA (22). In contrast, the extent of uracil in the nuclear DNA of the thyroid as detected with the UDG modification of the comet assay is not prominent, compared with spleen and lung. This finding also suggests that spontaneous cytosine deamination to uracil is not a dominant event in the thyroid. Therefore, the high frequency of C->T transitions in somatic mutations of the TSHR is unlikely to result from spontaneous deamination of cytosine. Again, an alternative explanation involves oxidation of cytosine to 5-hydroxy-cytosine followed by deamination and 5-hydroxy-uracil formation (33, 34).

Considering the difference of the SMR in the lacZ reporter model between thyroid and liver, we expected higher scores for thyroid DNA in the unmodified comet assay, compared with other organs. This comet protocol also detects strand breaks, and more than 90% of the lacZ mutants were size change mutants (data not shown) that could result from strand breaks. However, we detected a score in the thyroid that is not significantly different from lung and spleen but lower compared with liver. Such a finding could be explained by a much higher number of abasic sites in the DNA of liver, lung, and spleen that are also detected with this comet protocol and would cover a low number of strand breaks. Alternatively lacZ size change mutants could stem from unrepaired replication errors that are not detectable with the comet assay. However, we detected a higher mRNA expression of APEX1 in liver, which could reflect a compensating mechanism to more efficiently repair abasic sites in DNA and would therefore argue in favor of the first explanation. Moreover, the expression of APEX1 (also known as Ref-1 protein) is increased by TSH in the thyroid cell line FRTL-5 (35), which supports a role for DNA repair in relation to thyrocyte activity. Differences in the expression and activity of certain DNA repair enzymes could provide an explanation for a low score of the thyroid in the unmodified comet assay, compared with increased scores for DNA base adducts (e.g. endonuclease III and FPG modification). Such a hypothesis will be the subject of further studies.

Finally we used an antibody to 8-oxoguanine to detect 8-OHdG/8-OHG in tissue sections of thyroid, spleen, liver, and lung. Distribution of 8-OHdG/8-OHG staining is highly consistent with our hypothesis: 1) it is most prominent in the follicular cells near the lumen in which H2O2 is generated; 2) it appears stronger in the follicular cells, compared with spleen, lung, and liver cells; and 3) our semiquantitative evaluation of the staining perfectly correlates (r = 0.94; P < 0.05) to the results of the FPG modified comet assay that includes detection of 8-OHdG. Moreover, OGG1 mRNA expression that would repair 8-oxoguanine modifications (36) expectedly shows higher expression in lung an organ with pronounced oxygen exposure. However, a very similar level of mRNA expression of OGG1 in the thyroid, which is higher, compared with spleen and liver, further supports our oxidative DNA damage hypothesis. In addition, preliminary data suggest a reduced expression of OGG1 in follicular thyroid cancer and higher expression in Graves’ disease (Karger, S., and D. Führer, unpublished data).

In contrast to other organs (37, 38), we did not detect an age-dependent increase in the SMR. However, the comparison of our data with other organs at any age suggests that the mutation rate in thyroid at 8 wk is already higher than rates reached in any other organ, even at ages over 30 months. It might therefore be very likely that the mutation rate has reached a plateau. A possible reason for such a plateau could be oxidative stress, which is very likely the cause of the accumulation of mutations with age (39). Our data suggest that oxidative stress is also the cause for the higher mutation rate in the thyroid. At this high level, oxidative stress caused by aging might not add to the normal oxidative stress already present in thyrocytes or is neutralized by antioxidative or DNA repair activity.

Our transgenic model using the lacZ reporter might overestimate the mutation rate because transcription-coupled DNA repair (TCR) (40, 41) is excluded for the lacZ reporter, which is not transcribed in eukaryotes. However, the frequency of mutations in the TSHR gene, which shows a medium to high transcription level in the thyroid, suggests that TCR is not active enough to compensate mutagenic insults. Further studies are needed to elucidate whether TCR activity affects the neoplastic potential of the thyroid.

Our study gives a possible explanation for the contradiction that a slow proliferating tissue like the thyroid gland shows a high frequency of somatic mutations and tumor initiation. Interpretation of our data suggests that the strikingly high SMR in thyroid could be due to oxidative DNA damage caused by the specifics of thyroid hormone synthesis, which involves generation of free radicals and reactive oxygen. Our data from the comet assay (lower number of strand breaks and abasic sites) as well as 8-OHdG/8-OHG staining (single base modification) are compatible with the occurrence of single-base mutations and do not suggest a general genomic instability of thyrocytes. As reviewed in Valko et al. (42), this could be the result of the specific antioxidant status or the DNA repair capacity of thyrocytes. However, with additional insults (e.g. ionizing radiation), the frequency of malignant transformation could rise dramatically as seen in radiation-induced papillary thyroid carcinomas. Because radiation also increases 8-oxoguanine modification, both radiation and endogenous oxidative stress could synergistically lead to the initiation of thyroid cancer (43). A high frequency of somatic mutations already in the normal thyroid will prompt us to study mutagenesis in the thyroid challenged by either nutritional restriction (e.g. iodine deficiency), genetic defects (e.g. oncogene expression), or radiation.


   Footnotes
 
This work was supported by Grant 10-1575-Pa I from the Deutsche Krebshilfe and the Interdisziplinäres Zentrum für Klinische Forschung Leipzig, Faculty of Medicine, Universität Leipzig (Projects B20 and Z03).

Disclosure summary: all authors have nothing to declare.

First Published Online April 20, 2006

Abbreviations: APEX1, DNA-(apurinic or apyrimidinic site) lyase; DMSO, dimethylsulfoxide; FPG, formamidopyrimidine DNA glycosylase; IHC, immunohistochemistry; OGG, 8-oxoguanine DNA glycosylase; 8-OHdG/8-OHG, 8-hydroxydeoxyguanosine/8-hydroxyguanosine; SMR, spontaneous mutation rate; TBS, Tris-buffered saline; TCR, transcription-coupled DNA repair; TSHR, TSH receptor; UDG, uracil-DNA-glycosylase.

Received December 30, 2005.

Accepted for publication April 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Castro MR, Gharib H 2005 Continuing controversies in the management of thyroid nodules. Ann Intern Med 142:926–931[Abstract/Free Full Text]
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