Peroxidase and NADPH-Cytochrome C Reductase Activity During Thyroid Hyperplasia and Involution

KUNIHIRO YAMAMOTO AND LESLIE J. DEGROOT Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637

ABSTRACT. The regulation of iodination was in- TSH injection, whether expressed per mg gland vestigated in male rats during physiological altera- weight, per mg protein, or per fxg DNA, suggesting tions in thyroid function. Thyroid hyperplasia was enzyme induction and cellular hypertrophy. Involu- Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 produced by giving 0.01% PTU in drinking water or tion by T4 administration caused a decrease in injection of TSH (2 USP U/day); involution was thyroid weight, DNA content, and enzyme activity per induced after PTU treatment by giving 3 fig L-T4/ml gland. The main reason for the decrease in enzyme in drinking water. Increase in activity of thyroid activity per gland was a diminution of cell numbers. peroxidase and NADPH-cytochrome c reductase per During thyroid hyperplasia and involution, perox- gland exceeded gains in thyroid weight and DNA idase and NADPH-cytochrome c reductase activity content early in hyperplasia, but increased essen- is regulated by TSH. During the onset of TSH tially in parallel manner during chronic PTU treat- action, peroxidase and NADPH-cytochrome c re- ment. Enzyme activity per /xg DNA increased to ductase increase to a greater extent than thyroid 155% of control after 4 days of PTU treatment, weight and DNA content, suggesting preferential decreased to 138% on the seventh day, and was at enzyme synthesis in addition to cell hypertrophy. the control level after 21 days of treatment. Cellular (Endocrinology 95: 606, 1974) enzyme activity also increased after seven days of

HE activity of thyroid peroxidase adjusted to 7.4 with sodium phosphate buffer. T is augmented by chronic TSH treat- Other groups of rats were given 1 USP U TSH ment and is depressed by thyroxine treat- in 0.5 ml of saline by injection subcutaneously ment (1,2). TSH is also known to cause twice a day for 1 to 7 days. (The TSH was a gift hyperplasia of the thyroid (3). It is of of the National Institutes of Health.) For induction of thyroid involution, L-thyroxine (Sigma interest to study the change in the ac- Chemical Co.) 3 /ug/ml was added to drinking tivities of the enzymes involved in thyroid water containing 0.01% PTU on the 21st hormone synthesis to determine whether through the 30th days from the beginning of TSH can specifically induce these en- PTU treatment. The rats were anesthesized zymes. For this purpose, the activities of lightly with ether and sacrified by exsanguina- peroxidase and NADPH-cytochrome c re- tion. The thyroids from each group were ex- ductase, thyroid weight and DNA content cised, chilled, trimmed of connective tissue, have been determined during the de- weighed, and pooled. Each group of thyroids velopment of thyroid hyperplasia and in- was homogenized in modified Krebs-Ringer- 5 volution. phosphate buffer containing 10~ M KI and cen- trifuged at 105,000 x g for 1 hr. The pellets were washed once and resuspended in the same Materials and Methods buffer. Peroxidase activity was estimated by Male Charles River CD rats were fed Purina thyrosine iodinase assay. The assay system Chow. At the beginning of the experiment the contained participate protein (80-120 fxg), 0.5 average body weight was 100 g (85-120 g). To ml K-R-phosphate buffer at pH 7.0, 10 nmoles 131 induce thyroid hyperplasia, 0.01% propyl- KI, 1 fxCi I, 1 /Ltmole glucose, 0.1 mg glucose thiouracil (PTU) was added to the drinking oxidase (Type II, Sigma Chemical Co.) and 1 water for periods ranging from 1 to 41 days. The /Ltmole tyrosine (Sigma Chemical Co.), in 1 ml. pH of the drinking water containing PTU was Incubation was started by adding glucose oxidase and stopped with 2 drops of 2N HC1 after 20 min at 37 C. The percentage of iodide Received September 4, 1973. Supported by United States Public Health Service formed into iodotyrosine was assessed by count- Grant No. AM-13,377. ing radioactivity adsorbed by cation exchange 606 IODINATION AND THYROID HYPERPLASIA 607 resin (Fisher Scientific Co.) at pH 2.0 (4). NADPH-cytochrome c reductase assay system 5 consisted of participate protein (80-120 /xg), 0.4 o ml of 0.2M sodium phosphate buffer at pH 7.0, 1 O_ oOC /xmole KCN, 50 nmoles cytochrome c (type III o horse heart, Sigma Chemical Co.) and 100 nmoles NADPH (Sigma Chemical Co.), in 1 ml. KCN was added to inhibit any effects of

mitochondrial enzymes on reduction of cyto- Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 chrome c. The reaction, at room temperature, was started by adding NADPH and recorded by spectrophotometry at 550 m/i. DNA was determined in duplicate by a modified Schmidt- Thannhausser-Schneider method (5). One-tenth ml of 20% homogenate (w/v) from 100 mg 0.010-1 thyroid was made up to 1 ml in ice water, and (B) NADPH-cytochrome cy then precipitated and washed twice with 0.5 ml reductase of 0.6N perchloric acid (PCA). The pellet was incubated with 0.8 ml of 0.3N KOH for one hour at 37 C. The acid-insoluble precipitate obtained c E after alkaline hydrolysis was washed with 1.2N O" 0.005- PCA. After the pellet was extracted with 0) ethanol-ether (3:1, V:V), it was hydrolyized with =1 0.8 ml of 0.5N PCA for 15 min at 90 C to release free bases. DNA content was estimated by spectrophotometry at 265 m/u,. The unknown sample was compared to a standard curve 10 100 200 derived from aliquots of commercial purified fxq PROTEIN DNA (Sigma Chemical Co.) carried through the same procedure. Protein was determined in FIG. 1. Enzyme activities of peroxidase and NADPH- duplicate by the method of Lowry et al. (6). cytochrome c reductase as a function of enzyme protein concentration. Particulate fractions from non- stimulated thyroid (—•—) and hyperplastic thyroid Results after 41 days of PTU treatment ( A ) were used. It was necessary to pool thyroid tissue The assays were done as indicated in Materials and from groups of seven control animals in Methods. order to have sufficient material for each enzyme and DNA assay. Since the hyper- Change in thyroid weight and DNA con- plastic thyroids became about 5 times larger tent during treatment programs than those of the control group, dose re- Two days after introduction of PTU in sponse curves for both peroxidase and drinking water, thyroid weight increased NADPH-cytochrome c reductase assay significantly, doubling on the fourth day were made using particulate fraction from (Fig. 2, A). The increase in thyroid weight normal and hyperplastic thyroid in order to and DNA content per gland was linear validate the assay system (Fig. 1, A and B). until the 11th day. Thereafter, the rate of Both enzyme activities showed linearity of change decreased, although hyperplasia response over a range of 10-200 fig of still continued after 41 days of PTU treat- particulate fraction protein. Assays were ment. subsequently performed with nearly the TSH was injected subcutaneously (2 same amount of particulate protein (80-120 USP U/day) for 1 to 7 days. After 7 days, /ug/tube) from the control or treated there was a significant increase in thyroid thyroids in each test. weight (Fig. 3, A). "Involution" was 608 YAMAMOTO AND DEGROOT Enclo • 1971 Vol 95 • No 2

80- 300 FIG. 2. Changes of thyroid weight, DNA content, and activity of peroxidase and 50- h200 NADPH-cytochrome c reduc- Q tase during PTU treatment. A. o> Thyroid weight and DNA con- •100 tent. Thyroid weights (— • —) Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 are presented as a mean ± SD from 7 animals and DNA ( o ) is presented as an average of two samples. B. Per- centage change of peroxidase Q 6 < B and NADPH-cytochrome c re- ^_^ -*• o 400- ductase activity per gland and * Peroxidase DNA content per gland. En- Ul 0. o NADPH-cytochrome c reductose zyme activities are averages of f / A DNA 300- /1 / three samples and DNA is average of two samples. — • — J peroxidase per gland, —o — o 200- / o NADPH-cytochrome c reductase per gland, A DNA dl 100- & per gland. C. Percentage i change of specific activity. Specific activity of enzyme was 2001 estimated as activity per /ng • Peroxidase DNA. pecific activity per mg o o NADPH-cytochrome c reductase protei;. and per mg thyroid weight was almost the same as O per /xg DNA. — • — Perox- o 100 idase, — o— NADPH- cytochrome c reductase. 12 4 7 11 21 31 DAYS

achieved by T4-treatment of rats with idase and NADPH-cytochrome c reductase PTU-induced hyperplastic glands. The de- were parallel during all treatment pro- crease in thyroid weight was significant on grams. When rats were treated with PTU, the second day and was 50% on the 9th day the rate of increase of both enzyme ac- as compared with the group which re- tivities decreased after the 7th day of ceived PTU treatment alone for the same treatment. Between the 2nd day and the period (Fig. 4, A). DNA content per gland 11th day of PTU treatment, enzyme ac- also decreased, but after 6 days DNA tivities increased to a greater extent than content per weight of thyroid was higher DNA content (Fig. 2, B). TSH injection than in normal or hyperplastic thyroids. produced similar results (Fig. 3, B). Involu- tion caused by T4 administration resulted Change in the activities of peroxidase and in a greater decrease in enzyme activities NADPH-cytochrome c reductase during than in DNA content per gland (Fig. 4, B). treatments Specific activities of peroxidase and Both peroxidase and NADPH-cyto- NADPH-cytochrome c reductase chrome c reductase activity per gland increased during stimulation by PTU Specific activity of enzyme was esti- treatment or TSH injection and decreased mated as activity per fig DNA or per mg of during involution. The changes of perox- protein. After 2 days of PTU treatment, the IODINATION AND THYROID HYPERPLASIA 609

served on the 4th day and reached 160% of the control level on the 7th day of treat- 13- ment (Fig. 3, C). During involution, there -50 was a latent period of 4-5 days before < decreased enzyme activities per DNA were UJ o observed. On the 9th day, the specific 10- activities were 66% of the control level

(Fig. 4, C). Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 Enzyme activities per mg of protein or per mg of thyroid weight were also calcu- lated. With PTU treatment, enzyme activities per fj,g of DNA, per mg of weight, or per mg of protein, showed the same result.

80-. 300

--o •*——< 200 40-

100 200-

o r- Z 100 o o 100 so-

FIG. 3. Changes of thyroid weight, DNA content and activity of enzymes during TSH treatment. NADPH- cytochrome c reductase activity exactly paralleled 100 peroxidase. A. Change of thyroid weight (— • —) and DNA content ( o ), as shown in Fig. 2, A. B. 50- Percentage change of enzyme per gland and DNA Content per gland, as shown in Fig. 2, B. —• — peroxidase per gland, A DNA per gland. C. Percentage change of specific activity. Specific activities per /x.g of DNA (— • —), and per mg protein ( A ) are shown. Specific activity per mg weight was almost identical to specific activity per DNA. FIG. 4. Changes of thyroid weight, DNA content, and activity of enzymes during involution. NADPH- enzyme activity per (xg DNA increased to cytochrome c reductase varied exactly in parallel with peroxidase. A. Changes of thyroid weight (—• —) and 150% of control level and remained ele- DNA content ( o ) as shown in Fig. 2, A. B. vated through the 4th day (Fig. 2, C). Percentage change of enzyme activity per gland and Specific activity decreased to 138% on the DNA per gland, as shown in Fig. 2, B. —• — 7th day and returned to normal on the 21st peroxidase per gland, A DNA per gland. C. day. On the 31st and the 41st days, the Percentage change of specific activity. Specific activ- level was the same as the control. When ity per fig of DNA (— • —) and per mg of weight ( A ) are shown. Specific activity per mg of hyperplasia was induced by injection of protein was almost identical to specific activity per mg TSH, increased specific activity was ob- of thyroid weight. YAMAMOTO AND DEGROOT Endo • 1974 610 Vol 95 • No 2 When TSH was injected, the specific ac- tase, in order to investigate possible qual- tivities per fxg of DNA and per mg of itative dissimilarities of enzymes from weight were almost the same, but the glands with various degrees of stimulation. specific activities per mg of protein were Initial rates of reaction were measured at lower than the other parameters after the several concentrations of H2O2 (peroxidase) 3rd day of TSH injection. Involution re- or NADPH (reductase) and a fixed con- sulted in parallel decreases in the specific centration of iodide (peroxidase) or cyto-

activities per mg of weight and per mg of chrome c (reductase), using mitochondrial- Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 protein (Fig. 4, C). microsomal fraction (700 x g-105,000 x g pellet) of the thyroids from rats treated Kinetic study of peroxidase and NADPH- with PTU for 0, 4, or 30 days, respectively. cytochrome c reductase Lineweaver-Burke plots were constructed, relating the inverse of the reaction velocity Kinetic studies were performed using the to the inverse of the substrate concentra- triiodide assay of peroxidase (7), and stand- tion (Fig. 5). The same apparent Km was ard assay of NADPH-cytochrome c reduc- found with the three enzyme preparations, (A) Peroxidase indicating that both peroxidase and NADPH-cytochrome c reductase from •510 glands with various stages of stimulation had kinetic properties identical to control glands in the system studied. Discussion The present study was designed to measure changes in the activity of enzymes -5 10 thought to be involved in the thyroid 1/S mM) iodination system during development of

(B) NADPH-cytochrome c hyperplasia or involution. The develop- reductase "2 ment of thyroid hyperplasia produced by E 0.01% propylthiouracil was comparable to that observed by Wollman (3), who used 0.25% thiouracil for the treatment. In our data, the latent period before significant increase in thyroid weight was one or two days shorter than that reported by Woll- man. This may be due to differences in treatment or in the age of the rats. Possibly our younger rats have a higher susceptibil- -100 0 10 20 50 100 1/S (NADPH mM) ity to stimulation by TSH. A significant increase in rat plasma TSH levels within FIG. 5. Lineweaver-Burke plot of thyroid peroxidase 24 hr after 0.01% PTU treatment has been and NADPH-cytochrome c reductase relating the inverse of the reaction velocity to the inverse of the reported (8). substrate concentration. Mitochondrial-microsomal Peroxidase and NADPH-cytochrome c fractions of the thyroids from rats treated with PTU for reductase activities per gland increased 0 day (•—•), 4 days (A —A), and 30 days (o—o) during stimulation by TSH. Our data con- were used. Enzyme activity is expressed as the cerning the response of peroxidase to 7 change of O.D. at 353 m/u. per minute for triiodide assay, and as the change of O.D. at 550 rmj, per minute days of treatment with PTU, TSH, or T4 for NADPH-cytochrome c reductase assay, at the agreed with previous papers (1,2). Four initial rate of the reaction. responses of the iodination process to TSH IODINATION AND THYROID HYPERPLASIA 611 are suggested by our data and by other the 4th-5th day of TSH injection indicate reports: 1) iodide organification is rapidly the presence of cell hypertrophy, the third affected by changes in H2O2 generation type of response suggested above. without an increased level of peroxidase During PTU treatment, the specific activ- and NADPH-cytochrome c reductase (2,9), ity per fig of DNA and per mg of protein 2) initial increases in cellular enzyme ac- changed in parallel, indicating that the tivities are due to an induction of enzyme initial high cellular enzyme activity is

in preference to general cell protein syn- mainly due to specific enzyme induction in Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 thesis, 3) the cellular enzyme mass increases excess of general cell protein synthesis. as a consequence of cell hypertrophy, 4) Hypertrophy was not observed, possibly the major cause of increased enzyme ac- because during PTU treatment there may tivities per gland during chronic thyroid be rapid induction of hyperplasia by the hyperplasia is the proliferation of thyroid continuous high TSH levels. With a longer cells. period of PTU treatment where great During the first 3 days of TSH injec- hyperplasia was found (11 days or more), tion, neither peroxidase nor NADPH- the enzyme specific activity became nor- cytochrome c reductase activity changed. mal. This delineates the fourth response of This is consistent with previous data (10). thyroid to TSH; iodinating enzyme activity However, Rosenfeld and Rosenberg re- is finally regulated by total cell number in ported that TSH stimulated PBI formation the thyroid. This fourth response to TSH is in vivo three hours after injection under the major long-term response and may rep- conditions where iodide trapping might no resent a "balance state" in terms of enzyme longer be rate limiting (9). These early activity per cell. effects of TSH on organification might be Kinetic studies of peroxidase and due to the increased availability of reduced NADPH-cytochrome c reductase show that pyridine nucleotide for H2O2 generation. the enzymes from glands at various stages Ogata et al.t reported a prompt" change of of stimulation have the same apparent redox state of pyridine nucleotide in situ Km's. Thus the decline in enzyme activity caused by TSH (11). NADPH-cytochrome c per cell to basal levels with prolonged TSH reductase, NADPH, and cytochrome c may stimulation is due to reduced levels of produce H2O2 in the thyroid (12). enzyme rather than an alteration in quality Our observations after TSH injection of enzyme. indicate the presence of the second and the Data on the involution caused by T4 third responses of the thyroid to TSH. administration also demonstrate the several Many hormones are known to stimulate a mechanisms in the TSH response. During specific induction of enzyme in their target involution, enzyme activity per gland and organs (13). In this type of response, both per cell decreased. Specific enzyme activ- specific activity per fig of DNA and per mg ity depression was observed only on days of protein should increase. But if the 6-9, and evidence for decrease in cell size cellular enzymes increase in parallel with was present on days 4-9. However, the increased cell mass during hypertrophy, major diminution in enzyme activity is we would see increased specific activity caused by the decline in cell numbers, per DNA with unchanged specific activity because the specific activity of enzyme did per mg of protein. The second type of not decrease on the 4th day while thyroid thyroid response to TSH is indicated by weight, DNA content, and enzyme activity increased specific activity per mg of pro- per gland decreased by 60%. The low tein on the seventh day of TSH injection. thyroid weight per fig of DNA in the The greater increase of enzyme activity per involuted thyroid is consistent with data fig of DNA than per mg of protein during from Wollman (3) and Greer (14) and is YAMAMOTO AND DEGROOT Endo 1974 612 Vol 95 ( No 2 represented histologically by microfollicu- H2O2 generation defect, peroxidase activity lar cells. Specific activity of peroxidase and per mg of protein is reported normal (15). NADPH-cytochrome c reductase per mg of Also, in the Clayton type goiter in which an thyroid weight or per fig of DNA is below abnormal peroxidase is found, NADPH- the control level in the involuted gland. cytochrome c reductase level per mg of This indicates that TSH is important for protein is normal (16). These reports are maintaining a basal level of enzyme activ- consistent with our data.

ity in the thyroid. Downloaded from https://academic.oup.com/endo/article/95/2/606/2618917 by guest on 30 September 2021 Our data on involution of the stimulated References thyroid by T4 administration are very similar to those reported by Wollman (3). 1. Zimmerman, A. E., and C. C. Yip, Can J Physiol Thyroid weight and DNA did not return to Pharmacol 46: 449, 1968. 2. Nagataki, S., H. Uchimura, Y. Masuyama, and K. normal. Greer et al. also observed the same Nakao, Endocrinology 92: 363, 1973. phenomenon while inducing "colloid goi- 3. Wollman, S. H., and T. R. Breitman, Endocrinol- ters" in rat (14). The high DNA content ogy 86: 322, 1970. indicates that the thyroid enlargement pres- 4. DeGroot, L. J., S. Jaksina, and M. Karmarkar, Endocrinology 83: 1253, 1968. ent after involution is not only due to 5. Munro, H. N., and A. Fleck, Analyst 91: 78, 1966. colloid retention, but also to a greater 6. Lowry, H. O., N. J. Rosebrough, A. L. Farr, and R. number of cells. These results probably J. Randall J Biol Chem 193: 265, 1951. relate to the difficulty in reducing the size 7. Taurog, A., M. L. Lothrop, and R. W. Estabrook, of endemic goiters by hormone treatment Arch Biochem Biophys 139: 221, 1970. or iodide supplementation. 8. Griessen, M., and T. Lemarchand-Beraud, Endo- crinology 92: 166, 1973. Peroxidase and NADPH-cytochrome c 9. Rosenfeld, P. S., and I. N. Rosenberg, Endo- reductase changed their activities almost in crinology 78: 621, 1966. parallel during all treatments. This sug- 10. DeGroot, L. J., and A. D. Dunn, Endocrinology gests that these two enzymes may exist in 78: 1032, 1966. the same unit of the thyroid cell membrane 11. Ogata, E., K. Nishiki, S. Kobayashi, K. Tateishi, and H. Suzuki, Endocrinology 87: 552, 1970. structure, since peroxidase and NADPH- 12. Nagasaka, A., L. J. DeGroot, R. Hati, and C. Liu, cytochrome c reductase are membrane- Endocrinology 88: 486, 1971. bound in the thyroid. 13. Gelehrter, T. D., Metabolism 22: 85, 1973. There are a few papers reporting enzyme 14. Greer, M. A., H. Sruder, and J. W. Kendall, levels in human congenital goiters which Endocrinology 81: 623, 1967. were compensated by increased endoge- 15. Ljunggren, J. G., Ada Endocrinol 72: 272, 1973. 16. Hagen, G. A., H. Niepomniszcze, H. Haibach, M. nous TSH possibly for more than 10 years. Bigazzi, R. Hati, B. Rapoport, C. Jimenez, L. J. In Pendred's syndrome, in which the DeGroot, and T. F. Frawley, N Engl J Med 285: organification defect is probably due to an 1394, 1971.