J. Endocrinol. Invest. 15: 519-523, 1992
The effect of iodine on lipid peroxidation and ultrastructure in the thyroids of BB/Wor rats 1 E.M. Allen 2 University of Maryland Medical School, Baltimore VAMC, 3900 Loch Raven Blvd., Baltimore, MD 21218, USA ABSTRACT. There has been recent data suggesting that iodine potentiates lymphocytic thyroiditis (LT) by inciting oxidative stress. However, the mechanism by which iodine induces LT in genetically predisposed animals is unknown. This study was undertaken to examine LT -prone BBlWor (LT -P) rat thyroids for signs of acute iodine toxicity and oxidative damage before the onset of spontaneous LT. Lipid peroxidation was assessed by the measurement of malonyldialdehyde (MDA) in thyroid homogenates after randomization to a treatment group receiving 0.05% iodide in the drink-
ing water or tap water for 24 hours. Basal MDA levels were higher in LT-prone rat thyroids than Wistar rat thyroids, b,ut iodine treatment did not influence intrathyroidal MDA levels. Electronmicroscopy demonstrated that prolonged treatment with excess iodine, increased the number of apical Iysosomes. But there were no ultrastructural changes unique to LT-P rat thyroids. These data suggest that although LT -P rat thyroids may experience subclinical oxidative damage before the onset of histologically demonstrable LT, this activity is not affected by iodine.
INTRODUCTION
poration into thyroglobulin. Recent data suggest that iodide potentiates LT by inciting oxidative stress, Bagachi et al. have shown that antioxidants delay the onset of LT in the OS chicken (10). Similarly, the ability of the thioureas to retard LT may be through inhibition of the intrathyroidal oxidative processes necessary for iodide organification (11). The BioBreeding Worcester (BB/Wor) rat is an inbred strain of Wi star Furth rat which develops spontaneous lymphocytic thyroiditis and diabetes mellitus (12). In certain lines, the incidence of LT approaches 90% at 120 days of age, but LT rarely occurs before 90 days of age (13). Excess iodine ingestion accelerates the incidence of LTat 90 days of age from 30% to approximately 70% (2). These experiments were designed to examine the thyroids of LT-prone BB/Wor rats for signs of oxidative damage before the onset of spontaneous LT. One of the earliest signs of oxidative damage is lipid peroxidation. This reaction has been shown to occur in the liver, lung, kidney, brain and heart less than 24 hours after challenge with oxidizing agents such as bromobenzene (14). If a predisposition to oxidative stress is one of the genetic defects responsible for iodine-induced LT, then inflammation, lipid peroxidation and ultrastructural changes should occur in the thyroids of LT-prone animals after an iodine load. Any transient early inflammatory event could prime the system with self-antigen and thereby potentiate the development of LT.
Ingestion of excess dietary iodine is associated with lymphocytic thyroiditis (L T) in genetically predisposed dogs, rats, and chickens (1-4). However, the mechanism by which iodine precipitates LT is unknown. Cell necrosis and ultrastructural changes have been reported in the goitrogen-induced hyperplastic thyroids of rats and mice exposed to excess iodine (5-7). Follis examined the effect of 7.6 mg iodine, ip on hyperplastic hamster thyroids (89). Undoubtedly, the Wolff-Chaikoff effect limited iodine access into thyroid cells but was not protective because the iodine-exposed thyroids developed acute inflammatory changes within 24 hours. Therefore, excess iodine appears to have acute toxic effects in hyperplastic thyroids. The thyroid concentrates administered iodine in its inorganic form iodide (I). Iodide is a potent oxidizing agent which can be oxidized further by only H20 2 or O 2 , Within the thyroid, thyroid peroxidase oxidizes iodide in the presence of H2 0 2 for incor-
1Funded by the Veteran's Administration Merit Review Program. 2Formerly: Assistant Professor of Medicine. Meharry Medical College. Alvin C York VAMC. 3400 Lebanon Road. Murfreesboro, TN, USA. Key-words.' Iodine, lipid peroxidation, rat thyroid, thyroid ultrastructure Correspondence. E.M. Allen, MD, University of Maryland Medical School, Loch Raven VAMC, 3900 Loch Raven Blvd, Baltimore, MD, 21218, USA.
Received January 30,1992; accepted May 21,1992.
519
E.M. Allen
MATERIALS AND METHODS Experimental Animals
known concentrations of tetraethoxypropane (TEP) which converts quantitatively to MDA in 1: 1 stoichiometry. All samples were assayed in triplicate. Livers were assayed individually. However, the thyroids had to be pooled into groups of five. The results are expressed as nmol MDA / mg protein. The treatment groups are compared by AN OVA with Bonferroni correction.
Weanling 21-30 day-old NB line LT-Prone (LT-P) and WA line LT-Resistant (LT-Res) BB/Wor rats were obtained from the University of Massachusetts Medical Center breeding colony (Worcester, MA). Wistar Furth (Wis) rats were obtained from the Charles River BioBreeding Facility (Wilmington, MA). LT-P rats were tested for glucosuria twice weekly beginning at 56-60 days of age by using Tes-Tape (Eli Lilly Co., Indianapolis, IN). Animals with glucosuria were treated with 2-4 units of U-1 00 pure pork PZI insulin (Eli Lilly Co.) sc to maintain trace glucosuria and prevent weight loss.
Protein assay
Protein concentrations were measured according to Lowry (16). Histology
Ten six-week-old LT-P BB/Wor and 10 Wis rats were randomized to receive 0.05% iodide water overnight. The thyroids were removed, fixed in formalin, and subsequently stained with hematoxylin and eosin. These specimens were read by a pathologist for the presence of inflammatory changes characterized by follicular destruction and leukocytic infiltration. Electronmicroscopy was performed on the thyroids of LT-P BB/Wor, LT-Res BB/Wor and Wis rats which had been randomized at 30 days of age to receive either 0.05% iodide or tap water for 10, 30, or 50 days. At the end of that time, the thyroids were removed, and fixed in glutaraldehyde followed by osmium postfixation.
Treatment protocol
LT-P, LT-Res, and Wis rats were fed standard chow and randomized to receive either tap (controls) or 0.05% iodide (as Nal) water. Overnight, most rats ingested approximately 25 ml of water. Therefore, the treatment groups received nearly 12.5 mg of iodine, daily. At the end of each study, all of the animals were euthanized by cervical dislocation after ketamine anesthesia. At that time, the thyroids and livers were removed. MOA assay
Malonyldialdehyde (MDA) is an end-product of lipid peroxidation that can be measured as an index of oxidative damage. Although this assay is not highly specific for the detection of lipid peroxidation, it is very sensitive. Thirty LT-P BB/Wor rats and thirty Wi star rats between seven and eight weeks of age were randomized to receive 0.05% iodide or tap water for 24 hours. The MDA assay was performed on the thyroids and livers according to the method of Ohkawa et al. (15) as follows. After the excised tissue was washed in 0.9% NaCI solution, homogenates were prepared in a ratio of 200 mg wet tissue to 2.0 ml 1.15% KCI using a Ten Broeck manual homogenizer. To a sample of 0.2 ml tissue homogenate were added the following: 0.2 m18.1 % dodecyl sodium sulfate (8D8), 1.5 ml 20% acetic acid reagent, pH 3.5, and 1.5 ml 0.08% aqueous thiobarbituric acid (TBA). This mixture was brought to a final volume of 4.0 ml with distilled water, incubated at 90 degrees C for 60 minutes, then cooled in tap water. Five ml n-butanol:pyridine solution (15:1, v/v) and 1.0 ml distilled water were added to the homogenate and shaken vigorously. After centrifugation at 1,500xg for 10 minutes, the upper (organic) layer was separated and the absorbance read using a Turner 112 fluorometer (excitation 515 nm, emission 553 nm). The relative fluorescence is directly proportional to the MDA concentration. Working standards were prepared from
RESULTS
Basal intrathyroidal MDA levels were slightly higher in LT-P rats than Wis rats (pz 0
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Fig. 1 - The effect of overnight iodine on MOA in L T-Prone BB/Wor and Wistar Rats. p
The effect of iodine on lipid peroxidation and ultrastructure in the thyroids of BB/Wor rats 1 E.M. Allen 2 University of Maryland Medical School, Baltimore VAMC, 3900 Loch Raven Blvd., Baltimore, MD 21218, USA ABSTRACT. There has been recent data suggesting that iodine potentiates lymphocytic thyroiditis (LT) by inciting oxidative stress. However, the mechanism by which iodine induces LT in genetically predisposed animals is unknown. This study was undertaken to examine LT -prone BBlWor (LT -P) rat thyroids for signs of acute iodine toxicity and oxidative damage before the onset of spontaneous LT. Lipid peroxidation was assessed by the measurement of malonyldialdehyde (MDA) in thyroid homogenates after randomization to a treatment group receiving 0.05% iodide in the drink-
ing water or tap water for 24 hours. Basal MDA levels were higher in LT-prone rat thyroids than Wistar rat thyroids, b,ut iodine treatment did not influence intrathyroidal MDA levels. Electronmicroscopy demonstrated that prolonged treatment with excess iodine, increased the number of apical Iysosomes. But there were no ultrastructural changes unique to LT-P rat thyroids. These data suggest that although LT -P rat thyroids may experience subclinical oxidative damage before the onset of histologically demonstrable LT, this activity is not affected by iodine.
INTRODUCTION
poration into thyroglobulin. Recent data suggest that iodide potentiates LT by inciting oxidative stress, Bagachi et al. have shown that antioxidants delay the onset of LT in the OS chicken (10). Similarly, the ability of the thioureas to retard LT may be through inhibition of the intrathyroidal oxidative processes necessary for iodide organification (11). The BioBreeding Worcester (BB/Wor) rat is an inbred strain of Wi star Furth rat which develops spontaneous lymphocytic thyroiditis and diabetes mellitus (12). In certain lines, the incidence of LT approaches 90% at 120 days of age, but LT rarely occurs before 90 days of age (13). Excess iodine ingestion accelerates the incidence of LTat 90 days of age from 30% to approximately 70% (2). These experiments were designed to examine the thyroids of LT-prone BB/Wor rats for signs of oxidative damage before the onset of spontaneous LT. One of the earliest signs of oxidative damage is lipid peroxidation. This reaction has been shown to occur in the liver, lung, kidney, brain and heart less than 24 hours after challenge with oxidizing agents such as bromobenzene (14). If a predisposition to oxidative stress is one of the genetic defects responsible for iodine-induced LT, then inflammation, lipid peroxidation and ultrastructural changes should occur in the thyroids of LT-prone animals after an iodine load. Any transient early inflammatory event could prime the system with self-antigen and thereby potentiate the development of LT.
Ingestion of excess dietary iodine is associated with lymphocytic thyroiditis (L T) in genetically predisposed dogs, rats, and chickens (1-4). However, the mechanism by which iodine precipitates LT is unknown. Cell necrosis and ultrastructural changes have been reported in the goitrogen-induced hyperplastic thyroids of rats and mice exposed to excess iodine (5-7). Follis examined the effect of 7.6 mg iodine, ip on hyperplastic hamster thyroids (89). Undoubtedly, the Wolff-Chaikoff effect limited iodine access into thyroid cells but was not protective because the iodine-exposed thyroids developed acute inflammatory changes within 24 hours. Therefore, excess iodine appears to have acute toxic effects in hyperplastic thyroids. The thyroid concentrates administered iodine in its inorganic form iodide (I). Iodide is a potent oxidizing agent which can be oxidized further by only H20 2 or O 2 , Within the thyroid, thyroid peroxidase oxidizes iodide in the presence of H2 0 2 for incor-
1Funded by the Veteran's Administration Merit Review Program. 2Formerly: Assistant Professor of Medicine. Meharry Medical College. Alvin C York VAMC. 3400 Lebanon Road. Murfreesboro, TN, USA. Key-words.' Iodine, lipid peroxidation, rat thyroid, thyroid ultrastructure Correspondence. E.M. Allen, MD, University of Maryland Medical School, Loch Raven VAMC, 3900 Loch Raven Blvd, Baltimore, MD, 21218, USA.
Received January 30,1992; accepted May 21,1992.
519
E.M. Allen
MATERIALS AND METHODS Experimental Animals
known concentrations of tetraethoxypropane (TEP) which converts quantitatively to MDA in 1: 1 stoichiometry. All samples were assayed in triplicate. Livers were assayed individually. However, the thyroids had to be pooled into groups of five. The results are expressed as nmol MDA / mg protein. The treatment groups are compared by AN OVA with Bonferroni correction.
Weanling 21-30 day-old NB line LT-Prone (LT-P) and WA line LT-Resistant (LT-Res) BB/Wor rats were obtained from the University of Massachusetts Medical Center breeding colony (Worcester, MA). Wistar Furth (Wis) rats were obtained from the Charles River BioBreeding Facility (Wilmington, MA). LT-P rats were tested for glucosuria twice weekly beginning at 56-60 days of age by using Tes-Tape (Eli Lilly Co., Indianapolis, IN). Animals with glucosuria were treated with 2-4 units of U-1 00 pure pork PZI insulin (Eli Lilly Co.) sc to maintain trace glucosuria and prevent weight loss.
Protein assay
Protein concentrations were measured according to Lowry (16). Histology
Ten six-week-old LT-P BB/Wor and 10 Wis rats were randomized to receive 0.05% iodide water overnight. The thyroids were removed, fixed in formalin, and subsequently stained with hematoxylin and eosin. These specimens were read by a pathologist for the presence of inflammatory changes characterized by follicular destruction and leukocytic infiltration. Electronmicroscopy was performed on the thyroids of LT-P BB/Wor, LT-Res BB/Wor and Wis rats which had been randomized at 30 days of age to receive either 0.05% iodide or tap water for 10, 30, or 50 days. At the end of that time, the thyroids were removed, and fixed in glutaraldehyde followed by osmium postfixation.
Treatment protocol
LT-P, LT-Res, and Wis rats were fed standard chow and randomized to receive either tap (controls) or 0.05% iodide (as Nal) water. Overnight, most rats ingested approximately 25 ml of water. Therefore, the treatment groups received nearly 12.5 mg of iodine, daily. At the end of each study, all of the animals were euthanized by cervical dislocation after ketamine anesthesia. At that time, the thyroids and livers were removed. MOA assay
Malonyldialdehyde (MDA) is an end-product of lipid peroxidation that can be measured as an index of oxidative damage. Although this assay is not highly specific for the detection of lipid peroxidation, it is very sensitive. Thirty LT-P BB/Wor rats and thirty Wi star rats between seven and eight weeks of age were randomized to receive 0.05% iodide or tap water for 24 hours. The MDA assay was performed on the thyroids and livers according to the method of Ohkawa et al. (15) as follows. After the excised tissue was washed in 0.9% NaCI solution, homogenates were prepared in a ratio of 200 mg wet tissue to 2.0 ml 1.15% KCI using a Ten Broeck manual homogenizer. To a sample of 0.2 ml tissue homogenate were added the following: 0.2 m18.1 % dodecyl sodium sulfate (8D8), 1.5 ml 20% acetic acid reagent, pH 3.5, and 1.5 ml 0.08% aqueous thiobarbituric acid (TBA). This mixture was brought to a final volume of 4.0 ml with distilled water, incubated at 90 degrees C for 60 minutes, then cooled in tap water. Five ml n-butanol:pyridine solution (15:1, v/v) and 1.0 ml distilled water were added to the homogenate and shaken vigorously. After centrifugation at 1,500xg for 10 minutes, the upper (organic) layer was separated and the absorbance read using a Turner 112 fluorometer (excitation 515 nm, emission 553 nm). The relative fluorescence is directly proportional to the MDA concentration. Working standards were prepared from
RESULTS
Basal intrathyroidal MDA levels were slightly higher in LT-P rats than Wis rats (pz 0
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~
w
z is g
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Fig. 1 - The effect of overnight iodine on MOA in L T-Prone BB/Wor and Wistar Rats. p
