Necrosis and apoptosis coexist in the thyroid during goitre development and involution, but little is known about their respective causes. To test the possible role of free radicals, we analysed separately necrosis and apoptosis in male Wistar rats with depressed or normal antioxidant protection. Vitamin E-deficient and -sufficient rats were made goitrous with perchlorate in drinking water; involution was induced by repeated injection of NaI, without or with methimazole. Increase of thyroid malondialdehyde concentration and decrease of glutathione peroxidase activity confirmed the depressed antioxidant protection in vitamin E-deficient rats. Plasma thyroxine and TSH levels were not modified. Necrosis (swollen cells) and apoptosis (pyknotic cells) were quantified on histological sections. In vitamin E-sufficient rats, dead cells were very rare in control thyroids, increased 3-fold in goitre and still further during involution. Necrotic epithelial cells predominated in the goitre and their number declined after iodide supplementation, without or with methimazole. In contrast, the number of apoptotic cells and the caspase-3 activity were increased in goitre and further increased after involution, with two-thirds of pyknotic cells being observed in the interstitium. Apoptosis was prevented by methimazole. Vitamin E deficiency significantly increased total cell death and epithelial cell necrosis and induced the occurrence of much cell debris in the follicular lumen during involution, with no modification of the apoptotic reaction. These results show that the type of cell death is differentially regulated during goitre development and involution: necrosis is related to the oxidative status of the cells, while apoptosis comes with iodine-induced involution.
JF Mutaku, JF Poma, MC Many, JF Denef and MF van Den Hove
JF Mutaku, MC Many, I Colin, JF Denef and MF van den Hove
The effects of the vitamins dl-alpha-tocopherol, ascorbic acid and beta-carotene, free radical scavengers and lipid peroxidation inhibitors, were analyzed in male Wistar rats made goitrous by feeding a low iodine diet (< 20 micrograms iodine/kg) and perchlorate (1% in drinking water) for 4, 8, 16, and 32 days. Groups of control or goitrous rats received for at least 16 days before killing a diet containing 0.6% vitamin E (as dl-alpha-tocopherol acetate), 1.2% vitamin C (ascorbic acid) and 0.48% beta-carotene, either simultaneously (vitamin cocktail) or separately. This treatment led to a 5-fold increase of vitamin E in the thyroid gland, a 24-fold increase in the liver and a 3-fold increase in the plasma. In control rats, vitamin cocktail administration increased slightly the thyroid weight with little changes in thyroid function parameters. During iodine deficiency, administration of the vitamin cocktail or vitamin E alone reduced significantly the rate of increase in thyroid weight, and DNA and protein contents, as well as the proportion of [3H]thymidine labeled thyroid follicular cells, but not that of labeled endothelial cells. Plasma tri-iodothyronine, thyroxine, TSH levels, thyroid iodine content and concentration as well as relative volumes of glandular compartments were not modified. The proportion of necrotic cells rose from 0.5% in normal animals to about 2% after 16 days of goiter development. No significant protective effect of the vitamins was observed. These results suggest that these vitamins, particularly vitamin E, modulate one of the regulatory cascades involved in the control of thyroid follicular cell growth, without interfering with the proliferation of endothelial cells.
M.-C. Many, J.-F. Denef, S. Hamudi, C. Cornette, S. Haumont and C. Beckers
The effects of iodide and thyroxine (T4) on female mice fed a low iodine diet (LID) for 8 weeks were analysed by morphological, stereological and biochemical methods. Iodide was given at a dose of 10 μg/day (HID) or 1 μg/day (MID), either alone or together with daily injections of 1 μg T4 for 8 or 40 days.
With HID, the thyroid weight and the numbers of follicles and cells remained higher than in controls, although cell necrosis occurred. Colloid volume increased and iodine was stored within the gland: a colloid goitre with non-functioning follicles was produced. With MID, the glands resumed an almost normal appearance. With T4 and LID, progressive normalization occurred, but after 40 days thyroid weight and numbers of follicles and cells remained higher than in controls. Glandular iodine content slowly increased and reached control value. The proportions of 125I-labelled tri-iodothyronine (T3) and T4 in thyroglobulin were reduced. With T4 and HID, the glands resumed a normal appearance. Neither necrosis nor folliculoneogenesis was noted. The proportions of 125I-labelled T3 and T4 in thyroglobulin were reduced, but T3 and T4 serum levels were higher than with HID. With T4 and MID, a normal state was obtained as early as day 8. After 40 days the gland was morphologically and functionally inactive.
In conclusion, the association of T4 and iodide seems to be the best way to obtain a rapid and complete involution of thyroid hyperplasia. The administration of T4 prevents the deleterious effects of an excess of iodine on follicular cells, and causes the gland to enter a slow-functioning state.
J. Endocr. (1986) 110, 203–210
M-C Many, S Maniratunga, I Varis, M Dardenne, H A Drexhage and J-F Denef
The administration of a high iodide dose (HID; 10 μg/day) to goitrous mice is known to induce thyroid cell necrosis and inflammation, which, in most strains, is transient. In this study, we analyzed the effects of iodide in autoimmune prone non-obese diabetic (NOD) mice.
Control NOD mice fed a standard diet (MID; 1 μg I/day) or HID did not spontaneously develop thyroiditis. In NOD mice previously made goitrous, HID provoked thyroid cell necrosis and diffuse inflammation within 4 days. Inflammatory cells consisted of MHC-class II+ antigen-presenting cells, CD4+ T helper cells and CD8+ T suppressor/cytotoxic cells. After 96 days of treatment with HID, thyroiditis similar to Hashimoto's disease was obtained in 100% of the animals, with destruction of thyroid follicles, large clusters of T and B cells, and antithyroid antibodies in the plasma. When treating goitrous mice with MID, no cell necrosis was observed and no autoimmune thyroiditis was obtained. The early iodide-induced cell necrosis and inflammation may thus be considered as an important factor in the induction and persistence of autoimmune thyroiditis in individuals carrying a genetic susceptibility to autoimmune disease.
Journal of Endocrinology (1995) 147, 311–320
AC Gerard, JF Denef, MC Many, P Gathy, C de Burbure, MF van den Hove, F Coppee, C Ledent and IM Colin
Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves' disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression of growth- and vasoactive-related factors (fibroblast growth factor-2, transforming growth factor-beta, endothelin-1, vascular endothelial growth factor, nitric oxide synthase III), and with microcirculation expansion. Observations were made on digitalised images of histological serial sections. The nearest-neighbour method was used to distinguish between random or clustered distribution. PCNA-positive cells were both randomly and uniformly distributed in homogenous goitres from C57 black mice, and were clustered in tIssue areas identified as papillary and hyperplastic zones in heterogeneous goitres from Tg-A2aR mice. However, they were absent in the so-called compact cellular zones featuring resting cells. Moreover, whereas papillary and hyperplastic zones were highly vascularised, compact zones were nearly free of microvessels. Spatial distribution of dividing cells was positively correlated with the expression of growth-related factors. A similar pattern was observed in the thyroids of patients with Graves' disease. In accordance with the recent demonstration of the presence of angiofollicular units in the thyroid, these data strongly support the hypothesis that functional and mitotic properties of each single thyrocyte, likely to be responsible for growth heterogeneity of hyperplastic glands, may be adjusted at tIssue level by specific interactions with neighbour endothelial cells that, in turn, could alter the mitotic rate of thyrocytes through paracrine signals.
I M Colin, P L Selvais, T Rebai, D M Maiter, E Adam, M-F vandenHove, J-M Ketelslegers and J-F Denef
Endothelin-1 (ET-1) is a major vasoconstrictor peptide, first found in endothelial cells, and later in many other tissues, including the thyroid gland. We analysed the expression of the ET-1 gene in the rat thyroid gland and changes in ET-1 mRNA and peptide levels in goiter development and involution, two circumstances characterised by vascular changes.
Thyroid hyperplasia was induced in adult Wistar rats by feeding a low iodine diet (LID) supplemented with 0·25% thiouracil for 10 days, and LID alone for 2 further days (H.12 group). Involution was induced by injecting 100 μg iodide and refeeding a normal diet during 6 h, 12 h, and 24 h (I.6h, I.12h, 1.24 h groups). Rats fed a normal iodine diet were used as controls.
A specific 488 bp cDNA corresponding to the known sequence of pre-pro ET-1 was found by RT-PCR from RNA extracts in all thyroid experimental groups, as well as in lung and kidney which were used as positive controls. RP-HPLC analysis showed that ET-1 immunoreactivity eluted similarly as mature ET-1
During hyperplasia, ET-1 mRNA and peptide levels were increased 3·5- and 5-fold respectively. The relative volume of the vascular bed was more than doubled. During iodide-induced involution, the glandular ET-1 mRNA level remained elevated. The concentration of ET-1 peptide increased and was significantly greater at 12 h involution than in the H.12 group. At this time, the capillary reticulum reverted to individual capillaries and the vascular bed was significantly reduced.
These data demonstrate that the ET-1 gene is expressed in the rat thyroid gland and that the ET-1 mRNA and peptide levels are increased during thyroid hyperplasia and remain elevated during a phase of rapid iodide-induced involution. These data suggest that changes in ET-1 production may play a role in control of thyroid gland trophic regulation and vascularity.
Journal of Endocrinology (1994) 143, 65–74