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Article Excerpt
Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PB-and PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5'-deiodinase I (5'-DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein-coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress-related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression. Key words: excess iodide, gene expression, micro arrays, oxidative stress, phenobarbital, propylthiouracil, thyroid, Wnt signaling. Environ Health Perspect 113:1354-1361 (2005). doi: 10.1289/ehp.7690 available via http://dx.doi.org/[Online 12 May 2005]
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Thyroid cancer, a fairly uncommon form of cancer in the human population, has causal links to environmental radiation exposures (Meirmanov et al. 2003). Although there are multiple epidemiology studies that associate radiation exposure with thyroid cancer, there have been no studies to associate human thyroid cancer with environmental chemical exposures (Hard 1998). The human thyroid responds toxicologically to multiple anti-thyroid drugs, including excess iodide, propylthiouracil (PTU), and thionamides, but the progression to cancer after repetitive administration has not been observed (Hard 1998; Hill et al. 1998; Markou et al. 2001). In contrast, multiple antithyroid drugs administered to the rat have demonstrated an increase in thyroid tumors, including PTU, thionamides, and the hepatic enzyme inducer phenobarbital (PB) (Capen 1997; Hurley et al. 1998; McClain et al. 1988). Other xenobiotics, including several organic iodides such as amiodarone and erythrosine, have also been associated with thyroid tumor development in the rat. Many organic iodides alter rat thyroid homeostasis, causing thyroid hypertrophy and alterations in colloid that may potentially lead to thyroid tumors after chronic administration (Capen 1997; Hurley et al. 1998).
The mechanisms by which organic iodides induce thyroid toxicity are varied and may include excess iodide being released into the blood during xenobiotic metabolism, toxicity to the liver that alters thyroid hormone metabolism, and/or direct thyroid toxicity that inhibits the release of thyroid hormones into the circulation (Capen 1997; Hurley et al. 1998). In subchronic toxicology studies it has been difficult to predict whether early changes in thyroid pathology could lead to thyroid cancer in the rat after chronic administration of organic iodides. Excess iodide alone can be toxic to thyroid cells in culture and cause thyroid hypertrophy and changes in colloid in vivo in the rat model (Capen 1997; Vitale et al. 2000). It has also been reported that administration of excess iodide promotes thyroid tumor development in rats initiated with N-bis(2-hydroxypropyl)-nitrosamine (Kanno et al. 1992). However, iodide excess alone has not been sufficient in inducing follicular cell hyperplasia and thyroid tumors in rats but more commonly causes hypothyroidism (Backer and Hollowell 2000; Kanno et al. 1994). This is believed to be due partly to the escape from the Wolff-Chaikoff effect (acute inhibition of iodine organification) that is seen within days of exposure to excess iodide (Wolff and Chaikoff 1948). This escape phenomenon is associated with the down-regulation of the sodium iodide (NaI) symporter (NIS) thereby reducing the amount of inorganic iodine in the thyroid so that thyroxine ([T.sub.4]) and thyroid-stimulating hormone (TSH) secretions are returned to normal physiological levels (Eng et al. 1999).
Chronic elevation of TSH levels has been associated with an increased risk of thyroid tumors in the rat, which may be due, in part, to the high turnover of the circulating thyroid hormone triiodothyronine ([T.sub.3]) in this species compared with the lower [T.sub.3] turnover rate in humans (Capen 1997). Many antithyroid drugs and PB mediate their carcinogenic properties by elevating circulating TSH levels in the rat albeit by different mechanisms (Hood et al. 1999; McClain et al. 1988). Although PTU reduces thyroid hormone production by inhibiting thyroglobulin organification in the thyroid and inhibiting the peripheral conversion of [T.sub.4] to active [T.sub.3], PB reduces circulating [T.sub.4] by increasing its hepatic metabolism and excretion via glucoronidation. Reductions in thyroid hormone ([T.sub.4] and [T.sub.3]) by either mechanism causes an elevation in TSH that is sufficient in causing thyroid tumors in rats after prolonged exposure without any evidence of thyroid DNA damage (McClain 1992). Organic iodides may also increase TSH levels by similar mechanisms; however, it is unknown what contribution iodide excess has in their overall toxicity to the rat thyroid. To this end we have implemented biochemical, pathological, and molecular analyses to characterize the rat thyroid response to three model toxicants: excess iodide by using NaI a noncarcinogen, and the rodent thyroid carcinogens PB and PTU, in a modified 2-week endocrine battery (O'Connor et al. 2002). The goals of this study were to identify dose-dependent gene expression profiles induced by excess iodide in rats, determine whether gene expression profiles could be obtained that correlate with clinical and pathological end points in rats, and determine whether profiles are predictive of the carcinogenic potential of each chemical in rats.
Materials and Methods
In Vivo Studies
Adult male Crl:CD (SD)IGS BR rats, approximately 8 weeks of age, were treated with NaI, PB, and PTU for 14 consecutive days. NaI, PB, and PTU were purchased from Sigma Chemical Company (St. Louis, MO). Rats (n = 20/group) were dosed by oral garage with vehicle (water or 0.25% methylcellulose), NaI (0.1, 1, 10, or 100 mg/kg/day), PB (100 mg/kg/day), or PTU (10 mg/kg/day) at a dose volume of 5 mL/kg. NaI was dissolved in water, whereas PB and PTU were dissolved in methylcellulose. On day 15, all rats were euthanized by carbon dioxide anesthesia and exsanguination. Blood samples were collected from the inferior vena cava of each animal at necropsy to measure serum levels of TSH, [T.sub.4], [T.sub.3], and reverse [T.sub.3] (r[T.sub.3]). Terminal body, thyroid gland, and liver weights were recorded for the first 10 animals of each dose group. The thyroid gland and surrounding tissue from the first 10 animals of each dose group were processed for histopathological evaluation. A liver sample from the first five animals of each dose group was processed to measure 5'-deiodinase I (5'-DI) and uridine diphosphate-glucuronyltransferase (UDPGT) activity. Thyroid glands from the last 10 animals (five from methylcellulose group) from each dose group were removed and placed in RNALater (Ambion, Austin, TX) overnight at 4[degrees]C. The next day, thyroids were removed from the RNALater and stored at -80[degrees]C until processed for total RNA.
The research described in this publication was conducted in a laboratory accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International, and the investigators complied with the regulations and standards of the Animal Welfare Act and adhered to the principles of the Guide for the Care and Use of Laboratory Animals (National Research Council 1996).
Pathological Evaluations
After euthanization the thyroid glands and surrounding tissue from the first 10 animals from each group were removed and placed into formalin fixative for at least 48 hr before trimming and...
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