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Maier, Krohn, Paschke
Iodine deficiency activates antioxidant genes and causes DNA damage in the thyroid gland of rats and mice.
Maier J, van Steeg H, van Oostrom C, Paschke R, Weiss RE, Krohn K.
Biochim Biophys Acta. 2007 Mar 24; [Epub ahead of print]
[abstract only]
"Because thyroid nodules are frequent in areas with iodine deficiency the aim of this study was to characterise molecular events during iodine deficiency that could explain mutagenesis and nodule formation. We therefore studied gene expression of catalytic enzymes prominent for H(2)O(2) detoxification and antioxidative defence, quantified DNA oxidation and damage as well as spontaneous mutation rates (SMR) in mice and rats fed an iodine controlled diet. Antioxidative enzymes such as superoxide dismutase 3, glutathione peroxidase 4 and the peroxiredoxins 3 and 5 showed increased mRNA expression, which indicates increased radical burden that could be the cause of additional oxidized base adducts found in thyroidal genomic DNA in our experiments of iodine deficiency. Furthermore, the uracil content of thyroid DNA was significantly higher in the iodine-deficient compared to the control group. While SMR is very high in the normal thyroid gland it is not changed in experimental iodine deficiency. Our data suggest that iodine restriction causes oxidative stress and DNA modifications. A higher uracil content of the thyroid DNA could be a precondition for C-->T transitions often detected as somatic mutations in nodular thyroid tissue. However, the absence of increased SMR would argue for more efficient DNA repair in response to iodine restriction."
Deoxyribonucleic acid damage and spontaneous mutagenesis in the thyroid gland of rats and mice.
Maier J, van Steeg H, van Oostrom C, Karger S, Paschke R, Krohn K.
Endocrinology. 2006 Jul;147(7):3391-7. Epub 2006 Apr 20.
[abstract only]
"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."
Similar prevalence of somatic TSH receptor and Gsalpha mutations in toxic thyroid nodules in geographical regions with different iodine supply in Turkey.
Gozu HI, Bircan R, Krohn K, Muller S, Vural S, Gezen C, Sargin H, Yavuzer D, Sargin M, Cirakoglu B, Paschke R.
Eur J Endocrinol. 2006 Oct;155(4):535-45.
[abstract only]
"OBJECTIVE: Differences in iodine intake could account for the variable prevalences reported for somatic TSH receptor (TSHR) mutations in toxic thyroid nodules (TTNs). However, this question has not been settled, since no study has yet determined the TSHR mutation prevalence in regions with different iodine supplies in the same population using the same methodology. Therefore, we studied the prevalence of somatic TSHR mutations in TTNs from patients living in iodine-deficient or -sufficient regions in Turkey.
DESIGN AND METHODS: We screened 74 TTNs for somatic TSHR mutations. Exons 9 and 10 of the TSHR and 7 and 8 of the Gsalpha were screened by denaturing gradient gel electrophoresis. Determination of X-chromosome inactivation was used for clonality analysis.
RESULTS: TSHR mutations were identified in 52 (70.2%) of 74 TTNs. A Gsalpha mutation was identified in one TTN. Three new TSHR mutations were detected (A627V, I640K, I486N). No significant difference between frequencies of TSHR mutations in iodine deficient/sufficient regions was found. The frequency of non-random X-chromosome inactivation was similar in iodine-sufficient or -deficient regions and in TSHR mutation positive or negative hot nodules.
CONCLUSIONS: These findings suggest that TTNs in iodine deficient/sufficient areas predominantly arise from aberrant growth of a single cell. Our results suggest that neither the prevalence of TSHR mutations nor that of monoclonal TTNs is related to iodine supply."
Cold thyroid nodules show a marked increase in proliferation markers.
Krohn K, Stricker I, Emmrich P, Paschke R.
Thyroid. 2003 Jun;13(6):569-75.
[abstract only]
"Thyroid follicular adenomas and adenomatous thyroid nodules are a frequent finding in geographical areas with iodine deficiency. They occur as hypofunctioning (scintigraphically cold) or hyperfunctioning (scintigraphically hot) nodules. Their predominant clonal origin suggests that they result from clonal expansion of a single cell, which is very likely the result of a prolonged increase in proliferation compared with non-affected surrounding cells.
"To test whether increased cell proliferation is detectable in cold thyroid nodules, we studied paraffin-embedded tissue from 40 cold thyroid nodules and their surrounding normal thyroid tissue for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1 antibody) epitopes as markers for cell proliferation. All 40 thyroid nodules were histologically well characterized and have been studied for molecular characteristics before. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated.
"In 33 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for PCNA was detectable. In 19 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for Ki-67 was detectable. Moreover, surrounding tissues with lymphocyte infiltration showed a significantly higher labeling index for both PCNA and Ki-67 compared with normal surrounding tissue.
"These findings are first evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most cold nodules. However, the increase of proliferation markers shows a heterogeneity that is not correlated with histopathologic, molecular, or clinical characteristics."
Somatic mutations in thyroid nodular disease.
Krohn K, Paschke R.
Mol Genet Metab. 2002 Mar;75(3):202-8. Review.
[abstract only]
"Thyroid nodules can be found in up to 50% of inhabitants of iodine-deficient areas and are classified as hot or cold thyroid nodules according to their scintigraphic characteristics. Studies of hot thyroid nodules with comparable mutation detection methods and screening at least exon 10 of the TSH receptor reported frequencies for somatic TSH-receptor mutations ranging from 20 to 82% in patients with similar iodine supply. We have recently screened 75 hot thyroid nodules for somatic TSH-receptor mutations with the more sensitive DGGE method and found somatic TSH-receptor mutations in 57% and Gsalpha mutations in 3%. As 50% of the mutation-negative nodules from female patients are of monoclonal origin when tested for X-chromosome inactivation somatic mutations in other genes are likely to cause the development of hot thyroid nodules. Scintigraphically nonsuppressible areas have been identified in up to 40% of euthyroid goiters in iodine-deficient areas. We recently identified somatic TSH-receptor mutations in microscopic autonomous areas with increased 125T uptake in euthyroid goiters studied by autoradiography 20 years ago. These constitutively activating somatic TSH-receptor mutations in minute autoradiographically hot areas of euthyroid goiters are very likely starting foci which most likely lead to toxic thyroid nodules in iodine-deficient goiters. Therefore iodine deficiency does not only lead to euthyroid goiters but also to thyroid autonomy. The latter is also suggested by epidemiologic studies. Similar mechanisms induced by iodine deficiency and the subsequent hyperplasia, mutagenesis, and selection of cell clones could also lead to cold thyroid nodules by somatic mutations that only initiate growth but not hyperfunction of the affected thyroid epithelial cell. Somatic ras mutations have frequently been detected in histologically characterized thyroid adenomas or adenomatous nodules. However, they seem to be rare in cold thyroid nodules. Since the majority of these latter nodules and 60% of the cold thyroid nodules are monoclonal other somatic mutations are likely in these nodules."
Growth factor expression in cold and hot thyroid nodules.
Eszlinger M, Krohn K, Kratzsch J, Voigt C, Paschke R.
Thyroid. 2001 Feb;11(2):125-35.
[abstract only]
"Hot thyroid nodules (HTNs) are predominantly caused by constitutively activating thyrotropin receptor (TSHR) mutations leading to an activation of the cyclic adenosine monophosphate (cAMP)-cascade that stimulates growth and function of thyroid epithelial cells and confers growth advantage. In contrast to HTNs, the molecular etiology of scintigraphically cold thyroid nodules (CTNs) is largely unknown. An increased prevalence of toxic multinodular goiters in iodine-deficient regions has been reported. Growth factors increase during early stages of iodine deficiency in rats. These growth factors could modulate the proliferation of thyrocytes.
"In order to determine if and which growth factors could modulate the increase in thyroid epithelial cell proliferation in late stages of CTNs and HTNs we investigated epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and TGF-beta1 concentrations by enzyme-linked immunosorbant assay (ELISA) in CTNs (n = 7), HTNs (n = 9), and their normal surrounding tissue (ST). Insulin-like growth factor-1 (IGF-1) was determined in CTNs (n = 5) and HTNs (n = 10) and their surrounding tissues by radioimmunoassay (RIA).
"We found lower concentrations of all investigated growth factors and iodine in CTNs compared to surrounding normal tissues (ST). Only iodine showed a significant difference. Furthermore, we found significantly lower concentrations of EGF and TGF-beta1 concentration in HTNs compared to their STs. Differences of TGF-alpha and IGF-1 were not significant.
"In conclusion, low EGF, TGF-alpha, and IGF-1 concentrations in most CTNs in spite of low iodine concentrations argue against a pathophysiologic role of EGF, TGF-alpha, or IGF-1 in late stages of CTNs. The low EGF, TGF-alpha, and IGF-1 concentrations in HTNs irrespective of their clonal origin or the presence or absence of activating mutations argue for increased cAMP as the primary cause for thyroid epithelial cell proliferation in established HTNs. However, the pathophysiologic significance of low TGF-beta1 concentrations in CTNs and HTNs remains to be elucidated. It might be possible that growth factors like EGF, TGF-alpha, TGF-beta1, and IGF-1 play a more prominent role during early clonal expansion and that aberrant intrinsic signaling through a somatic mutation (e.g., TSHR for HTNs) confers the predominant selective growth advantage in later stages of HTNs or CTNs."