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Thyroid hormones in human tumoral and normal nervous tissues1
Brain Research 801 Ž1998. 150–157
Research report
Thyroid hormones in human tumoral and normal nervous tissues Rosa Marıa ´ Calvo a
a, )
1
, Jose Marıa ´ Roda b , Marıa ´ Jesus ´ Obregon ´ a , Gabriella Morreale de Escobar
a
Unidad de Endocrinologıa del CSIC and Facultad de Medicina, UAM, Madrid, Spain ´ Molecular, Instituto de InÕestigaciones Biomedicas ´ b SerÕicio de Neurocirugıa, ´ Hospital La Paz, Madrid, Spain Accepted 26 May 1998
Abstract We have studied T4 and T3 concentrations, DNA and protein concentrations and 5X and 5 deiodinases in samples of brain tumors obtained at surgery from 49 patients, and, in most cases, also from surrounding normal tissue. T4 concentrations in normal cortical tissue Ž6.19 " 0.45 ngrg. were lower than in white matter, but the difference disappeared when referred to the DNA content Ž2.26 " 0.27 ngrmg DNA.. No other differences were found between cortical and white matter, or among cortical lobes. T4 in normal tissue was higher than previously reported, mostly from autopsy samples, whereas T3 Ž0.99 " 0.07 ngrg. was similar. 5X D-I activity was negligible as compared to 5X D-II Ž8.11 " 1.09 fmolrhrmg protein.. When expressed in relation to the different DNA contents of normal vs. tumoral tissue, 5X D-II activities were the same for both. 5D activity was highly variable in the tumoral tissue, with negligible activities in meningiomas and pituitary adenomas. When referred to the DNA content, T4 and 5X D-II were the same, but T3 concentrations were lower in the tumor Ž0.24 " 0.03 ngrmg DNA. as compared to normal Ž0.35 " 0.04 ngrmg DNA. tissue samples. Whether or not this decrease of T3 affects the expression of T3-sensitive processes remains to be studied. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Thyroxine; Triiodothyronine; Deiodinase; Human; Brain; Tumor
1. Introduction The thyroid hormones, L-thyroxine ŽT4. and 3,5,3X-triiodo-L-thyronine ŽT3., are not only essential for normal brain maturation, but also for normal brain function throughout life w17,26x. Most of the information on the concentrations of T4 and T3 in adult brain, the activities of the iodothyronine deiodinases involved in outer Ž5X D-I and 5X D-II. and inner ring Ž5D. deiodination, the local generation of T3 from T4 and the regulatory mechanisms maintaining tight homeostatic control of cerebral T3 levels has been obtained in the rat w7,12,13,25x. The possible involvement of thyroid hormones in the response to treatment of patients with severe depression w4,18,20x has renewed the interest in cerebral thyroid hormone levels and metabolism in the adult brain. Although the iodothyronine concentra-
) Corresponding author. Instituto Investigaciones Biomedicas, Arturo ´ Duperier 4, 28029 Madrid, Spain. Fax: q34-1-5854587; E-mail: [email protected] 1 Presented at the 22nd Annual Meeting of the European Thyroid Association, Viena, Austria, 1994.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 7 6 - 9
tions and the deiodinase activities in the developing human cerebral cortex of aborted fetuses have been reported w5,14,21x, there is very little information on the adult human brain because of the obvious difficulties involved. The thyroid hormone concentrations in brain and other tissues obtained from victims of sudden accidental death and from patients who died after fatal illness have been reported; however, the time elapsed between death and autopsy was up to 26 h w2x. More recently, another study w9x has also investigated the iodothyronine levels in nontumoral nervous tissue obtained during surgery or at autopsy Žwith delays of up to 72 h.. Regarding the monodeiodinating enzymes, there is a report on 5D in human brain tumors w23x and another on the biochemical properties of 5X D-II and 5D in normal human central nervous system ŽCNS. as measured in vitro w9x. We have measured the concentrations of T4 and T3, and the activities of 5X D-I, 5X D-II and 5D in samples from cerebral tumors and, whenever ethically possible, from an aliquot of the sample of the presumably normal tissue taken for morphological examination. In addition, we have compared the above parameters among normal cortical lobes and between normal cerebral cortex and white matter, as well as between tumoral and normal tissue.
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2. Materials and methods 2.1. Patients and tissue sampling In the first part of the study, 35 patients have been involved, 15 men and 20 women from the Hospital La Paz, Madrid. Their ages ranged from 15 to 78 years Ž48.4 " 2.7.. One patient was operated on for the removal of a gliosis and the rest for the removal of brain tumors of different types: 10 astrocytomas of all histological grades, eight glioblastomas, four meningiomas, two oligodendrogliomas, two oligoastrocytomas, two metastases and a ganglioglioma, a glioma, a neurinoma, a cordoma, a hamartoma, and an adenoma of the anterior pituitary. The patients were not known to be affected by any thyroid disorder. They were on dexamethasone Ž16 mgrday; 4 mg every 6 h. for 3 to 5 days before surgery. The patients were fasted overnight, and the tissue samples were obtained after 1–2 h of endotracheal anesthesia. Specimens of normal brain tissue, either preferably cerebral cortex or white matter were obtained in lobectomized brains; to minimize the influence of edema, they were taken from the zone most distal to the periphery of the tumor. An aliquot of the ablated tumor, and of the normal tissue were rapidly frozen in liquid nitrogen and kept at y708C until the determinations were carried out. Samples available for the present study weighed from 31 to 880 mg. The amount of tissue was often too small to perform all the analytical determinations. None of the patients had been treated by radiotherapy before the operation. In the second part of the study, tissue samples from another 14 patients were collected. Of these subjects, seven were female and seven were male with ages ranging from 38 to 77 Ž55.3 " 2.9.. The tissue samples were obtained from four meningiomas, two neurinomas, one astrocytoma, four glioblastomas, two pituitary adenomas and one aliquot of normal temporal cortex tissue obtained at surgery for epilepsy. In most cases Ž45 patients. a blood sample was obtained from the forearm during the operation, for the determination of plasma T4, T3, rT3, free T4 and thyrotropin ŽTSH. levels. The above parameters were also assayed in the plasma from 45 healthy volunteers of both sexes and similar ages working at our institute to obtain a normal range in a control population. All the investigations were performed in accordance with the guidelines in the Declaration of Helsinki and approved by the Ethics Committee of the Hospital La Paz, Madrid.
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as described elsewhere in detail w27x. In brief, the sample is homogenized directly in methanol, and w131 IxT4 and w125 IxT3 are added to each sample as internal tracers for recovery calculations. These tracers are added in amounts small enough to avoid interferences in the final RIAs. Appropriate volumes of chloroform are added to the extract with chloroform:methanol Ž2:1, v:v., twice, followed by backextraction into an aqueous phase, also twice. The aqueous phase from each back-extraction is pooled and further purified on Bio-Rad 1 = 2 resin columns, from which the iodothyronines are eluted with 70% acetic acid. The eluates containing the labeled tracers are pooled, evaporated to dryness and extensively counted for the calculation of individual recoveries. RIA buffer is then added, and the T4 and T3 contents determined by RIAs in triplicate at two dilutions. For the determination of rT3 we used the same procedure as for T4 and T3, but using w125 IxrT3 as recovery tracer. The limits of detection are 2.5 pg T4, 0.7 pg T3 and 1 pg rT3rtube. The molar cross-reactivities for the RIAs and the inter and intraassay variations Žbelow 10%. have been previously described w16,28,29,32x. 2.3. Determination of deiodinase actiÕities 5X D-II activity was assayed in normal and tumoral brain samples, as described w30x. Before each assay w125 IxrT3 or w125 IxT4 were purified by paper electrophoresis to separate the contaminating iodide. 5X D-II activity was assayed incubating 50–200 mg protein in 100 mM potassium phosphate buffer ŽpH s 7.0., 1 mM EDTA with approximately 80,000 cpm of w125 IxrT3, 2 nM rT3, 20 mM DTT, in the presence or absence of 1 mM 2-N-propyl-6-thiouracil ŽPTU. for 1 h at 378C. The total volume was 100 ml. The activity which was not inhibited by PTU was considered as 5X D-II, and the PTU-sensitive activity as 5X D-I. Some samples Ž n s 11. were also assayed using w125 IxT4, 2 nM T4 q 1 mM T3 and 20 mM DTT, as previously reported w30x. The 125 Iodide release was separated by ion-exchange chromatography on Dowex-50W-X2 columns equilibrated in 10% acetic acid. The production of equal amounts of iodide and 3X ,3-T2 was checked in some assays. The protein content was determined by the method of Lowry et al. w22x, after precipitation of the homogenates with 10% TCA to avoid interferences from DTT in the colorimetric reaction. 5D activity was measured as described w8x by incubating 40–50 mg protein in 100 mM potassium phosphate buffer ŽpH s 7.4., 1 mM EDTA with approximately 50,000 cpm of inner-ring labeled 5-w125 IxT3, 25 nM T3, 20 mM DTT and 1 mM PTU for 1 h at 378C.
2.2. Determination of T4, T3 and rT3
2.4. Other determinations
T4 and T3 were determined by highly sensitive and specific radioimmunoassays after extensive extraction and purification of the iodothyronines from tissues or plasma,
The concentration of DNA and protein were also determined in aliquots of the tissue homogenates by the methods of Burton w6x and Lowry et al. w22x, respectively.
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Hemoglobin content was also measured in the tissue samples, as described by Crosby and Furth w11x, to assess the possible contribution of the thyroid hormones contained in blood trapped in the tissue sample. As T4 and T3 were measured in serum, a mean value of 50% was assumed for the hematocrit in order to calculate this possible contribution. Serum TSH was assayed by the Dynotest TSH kit ŽHenning Berlin, Berlin, Germany.. The lower limit of sensitivity is 0.16 mIUrml. Free T4 was measured using the Two-Step Gammacoat Free T4 RIA kit from Clinical Assays ŽBaxter, Cambridge, MA.. 2.5. Drugs and reagents T4, T3 and 3,5-diiodo-thyronine Ž3,5-T2., PTU and were from Sigma ŽSt Louis, MO.. Reverse T3 ŽrT3. and 3X ,3-diiodothyronine Ž3X ,3-T2. were from Henning Berlin, Bio-Rad 1 = 2 resin from Bio-Rad Laboratories ŽRichmond, CA.. High specific activity w125 IxT3, w125 IxT4, w125 IxrT3, 131 w IxT4 Ž3000 mCirmg. were synthesized by us, as described w27x. Inner-ring labeled 5-w125 IxT3 Ž80 mCirmg. was used as substrate for 5-deiodinase. It was provided by Drs. R. Thoma and H. Rokos from Henning Berlin. DL-dithiothreitol
2.6. Statistical analysis Results are shown as mean values " S.E.M. Significance of differences between groups was measured by Student’s t-test when only two groups were compared, and by one-way analysis of variance in the case of three or more groups. All calculations, including correlations, were performed as outlined by Snedecor and Cochran w31x.
3. Results 3.1. Circulating parameters of thyroid hormone status The concentration of T4, T3, rT3, TSH and circulating free T4 were measured in the plasma of these patients, as well as in a group of healthy adults. Total plasma T4 levels were normal whereas total plasma T3 concentrations were decreased when compared to those of healthy human beings, as all of them were below the normal range ŽTable 1.. Plasma rT3 in the patients with brain tumors fell in the Table 1 Mean values Ž"S.E.M.. of T4, T3, rT3, TSH and FT4 in the plasma obtained from patients bearing brain tumors ŽA.; and the normal range of control adults values obtained from 45 healthy volunteers ŽB. T4 Žmgrdl. T3 Žngrdl. rT3 Žngrdl. TSH ŽmIUrml. FT4 Žngrdl. A 7.6"0.4 B 3.1–7.8
58.3"2.7 137–293
21.2"1.8 20–82
1.0"0.2 0.5–2.6
1.7"0.2 1.4–2.8
lower range of normal people. Results of circulating TSH and free T4 were also within the normal range of our control values. 3.2. Normal cerebral tissue 3.2.1. Cerebral cortical tissue Õs. white matter In presumably normal cerebral tissue, we did not find any statistically significant differences between the concentrations of thyroid hormone, deiodinases, DNA and protein, in samples from the cerebral cortex and underlying white matter, except for the concentration of T4 which was higher in white matter when expressed per gram of wet tissue ŽTable 2.. This difference was also found when the data were calculated only with paired tissue samples, namely, those obtained from the same patient. However, it was no longer found when the concentration of T4 was referred to DNA content instead of weight of the sample. 3.2.2. Cortical lobes of the brain We determined the same parameters as above in different areas of the cerebral cortex: frontal, parietal, occipital and temporal lobes of presumably normal tissue. We did not observe any significant change between areas in the concentrations of T4, T3 and DNA and in deiodinase activities. Only the protein concentration was lower in the parietal lobe of the cortex Ždata not shown.. 3.3. Tumoral Õs. normal cerebral tissue Fig. 1 shows changes in concentrations of DNA and protein and in the proteinrDNA ratio in tumoral vs. normal brain paired samples. As may be seen, DNA increased whereas the protein and proteinrDNA ratio decreased in malignant vs. normal cerebral tissue. T4 and T3 levels and 5X D-II activities are presented in Fig. 2. Hemoglobin was measured to assess the possible contribution to the tissue concentration of thyroid hormones which were actually contained in trapped blood. The results shown in Fig. 2 were not affected, because in all cases, except one, the hormone levels in the trapped blood represented less than 1% of the value. T4 concentration was 1.8 times higher in tumoral as compared to normal tissue, but the change disappeared when values were referred to the DNA content. On the contrary, there was no difference in T3 levels when expressed per gram of wet weight of the tissue, but when referred to the DNA content, there was a decrease to about 70% of the normal values. 5X D-II activity showed a pattern similar to that observed for the T4 concentration: high when expressed per gram of weight and the same when referred to DNA content. Fig. 3 shows the concentrations of T4 and T3 and the 5X D-II activity in the different types of brain tumors. The T4 concentration ranged from 45.1 ngrg in one of the
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Table 2 X Concentrations of T4, T3, DNA and protein, as well as type II 5 D activity, in samples obtained from cortical cerebral tissue as compared to underlying white matter
ng T4rg ng T3rg X 5 D-II Žfmolrhrmg protein. mg DNArg mg proteinrg ng T4rmg DNA ng T3rmg DNA X 5 D-IIrmg DNA Žfmolrhrmg DNA.
Cortical tissue
White matter
t statistic
6.19 " 0.45 Ž22. 0.99 " 0.07 Ž26. 8.11 " 1.09 Ž21. 3.0 " 0.2 Ž25. 92.8 " 4.6 Ž22. 2.26 " 0.27 Ž22. 0.39 " 0.05 Ž25. 243 " 35 Ž21.
11.89 " 2.21 Ž7.)) 1.17 " 0.11 Ž6. 9.16 " 1.52 Ž5. 3.8 " 0.6 Ž5. 100.8 " 11.6 Ž8. 2.53 " 0.40 Ž6. 0.51 " 0.17 Ž7. 374 " 120 Ž5.
P - 0.005 n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s., not significant. Values are means" S.E.M. The numbers in parentheses indicate the number of samples involved.
In order to explore the presence of 5X D-I deiodinase, in the first set of brain samples we have determined the activity of the 5X-D enzymes using w125 Ix-rT3 as substrate. We found that 5X D-I activity Žnot shown. was very low both in tumoral and normal tissues, with undetectable values in 50% of the samples. Even when the activity was
astrocytomas to 1.6 ngrg in the hamartoma. The highest concentration of T3 Ž3.5 ngrg. was found in an adenoma of the anterior pituitary whereas two glioblastomas showed the lowest level of T3 Ž0.23 ngrg.. 5X D-II activity ranged from 657 fmolrhrmg protein in the adenoma to 0.64 fmolrhrmg protein in one of the meningiomas.
Fig. 1. DNA and protein concentrations, and proteinrDNA ratio Žmean " S.E.M.. in paired samples from brain tumors and surrounding normal tissue. )Identifies a statistically significant difference Ž p - 0.05..
X
Fig. 2. Concentrations of T4 and T3 Žexpressed per gram of wet weight. and 5 D-II activity in normal and tumoral samples from human brain Ždotted bars.. Darker bars represent the same parameters referred to DNA content. )Identifies a statistically significant difference in tumoral vs. normal brain samples Ž p - 0.05.. Data are means" S.E.M. The numbers in circles are the number of observations.
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detected, in the other 50% of the samples 5X D-I represented less than 10% of the total 5X D activity. In view of the very low PTU-sensitive 5X D activity, further samples were assayed using T4 as the preferred substrate for this assay. Moreover, rT3 was also used in parallel, in order to validate the results of the first set of samples. The enzyme was determined in crude homogenates; no microsomal fractions or kinetic studies could be performed because of the very small amount of tissue available. Results obtained were comparable. Both were correlated positively Ž n s 11; y s 0.353 x q 4.525, r s 0.833; with the 5X D-II activity using T4 in the abscissa and the activity using rT3 in ordinates.. The means obtained were 9.01 " 2.43 and 12.5 " 3.76 fmolrhrmg protein, respectively, when w125 Ix-rT3 or T4 were used. 5D activity was also determined in these second brain tumoral samples and in four of the previous set ŽFig. 4.. Of
Fig. 4. 5D-III activity in brain tumors of different types, as well as in two samples from normal surrounding cortex ŽCx.. The identification of tumoral samples is the same as in Fig. 3.
17 tumors, only 13 contained 5D activity, the mean " S.E.M. being 40.33 " 17.16 fmolrhrmg protein. The tyrosyl ring deiodinase activity was also assayed in two normal cortical samples Ž226.4 " 14.65 fmolrhrmg protein.. Only two tumors Žone astrocytoma and one glioblastoma. showed higher 5D activity than that of the normal tissue samples. Meningiomas Ž n s 5. either showed no or very low inner ring deiodinase activity. Two adenomas of the anterior pituitary exhibited no 5D activity. No clear correlations were found for the concentration of T4, T3 or the deiodinase activities with age, sex, type of tumor or degree of malignancy.
4. Discussion Thyroid hormones play an essential role in brain development and function throughout life. Most evidence has been obtained from studies in animal models, and information in humans is scarce. In our study T4, T3, rT3 and 5X and 5-monoiodinases were determined in tumoral and normal tissue from different zones of the CNS obtained from living patients at surgery. 4.1. Normal cerebral tissue
Fig. 3. Concentrations of T4 and T3 Žexpressed per gram of wet weight. X and 5 D-II activity in different types of brain tumors. The identification of tumors is as follows: As: astrocytoma, Gb: glioblastoma, Me: meningiomas, Mt: metastases, Oa: oligoastrocytoma, Od: oligodendroglioma, Ne: neurinoma, Co: cordoma, Ha: hamartoma, Gl: glioma, Gg: ganglioglioma, Ad: adenoma.
Deiodinase activities were measured in normal brain samples, practically all the phenolic ring deiodinase activity present appears to be 5X D-II. To our knowledge these are the first results of 5X D-II activities in the four lobes of the cortex from human adults, as only data for the temporal lobe have been published previously w9x. 5X D-II activity has been reported in human fetal cortex w21x. Although 5X D-I has been reported in the rat w34x, the activity of 5X D-I activity in our human tissue samples was negligible. This is in agreement with Campos-Barros et al. w9x who did not find a PTU-sensitive 5X-deiodination, also suggesting the absence of 5X D-I activity in the adult human brain. Thyroid hormone concentrations were also determined in normal brain tissue but we did not find differences in
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either T4 or T3 concentrations among the four lobes of the cortex. This is in agreement with a recent report w9x, although thyroid hormones were only studied in the temporal and frontal lobes of individuals at autopsy or surgery. This might suggest that there is not a specific pattern of distribution of the iodothyronine levels in the lobes of the cerebral cortex, however, it should be pointed out that we have grouped the tissue samples according to a rough localization, which may not be representative of the whole lobe. In addition, we cannot exclude from present results that there is no region-specific distribution of T4 and T3 in the rest of the brain. In the rat we have shown higher T4 levels Žin ngrg. in cerebellum and thalamus and high T3 in thalamus, but both hormones are consistently distributed across other brain areas, cortex included w19x. Cortex T3 levels are comparable to those reported for the rat w10,19,33x and also to those reported in human brain samples obtained intraoperatively w9x, from victims of sudden death w2,9x and from human fetuses w21x. Tissue T4 levels are comparable to those found in developing fetal human brain w21x, but are 2.5 to 4.5 times higher than in the rat w10,19,33x. Our T4 values are also higher than that reported in two studies for victims of accidental death but the time elapsed between death and autopsy was up to 26 h w2x, and 72 h w9x, respectively. During this time, some degradation of T4 and T3 might occur as the 5D enzyme is active in tissues obtained postmortem w9x. T4 concentration in normal brain samples obtained at tumor excision or at resection in living patients was also clearly lower than that we report here, but only four samples were studied w9x. The possible cause for the difference among reports in the concentration of T4 has not been clarified. Hemoglobin was measured in the brain samples of our study to determine if trapped blood could account for the higher intracerebral T4 levels; however, the contribution was less than 1%. We have compared the ratio between the brain T4 and the total serum T4 of the present patients and that obtained by us in the adult rat w24x, and it is more similar Ž0.09 and 0.08, respectively. than that obtained for autopsied humans Ž0.035. w2x. We believe that present concentrations of T4 and T3 in normal brain tissue from patients with a brain tumor are likely to be more similar to those of normal people than values obtained at autopsy, but we cannot exclude other possible factors considering that our patients had low circulating T3. Factors which might be affecting the values found by us are the fact that the patients had a brain tumor and that the samples were obtained after a few days on corticosteroids and while the patient was undergoing surgery, involving anesthesia, all of which are not physiological circumstances. Malignancy has been observed to influence thyroid status, with alterations typical of non-thyroidal illness, where the most common findings are a decreased circulating T3 and an increased rT3, a normal T4 and FT4, without a compensatory increase in TSH. Most data were
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obtained from patients with lymphoma, breast and lung cancer, and carcinoma of the colon. We are not aware of studies on patients with brain tumors. In the present series we have not found an increase in rT3, but rather the opposite. Findings in patients with high glucocorticoid levels or after treatment with this type of steroids present both a low T3 and T4, and increased rT3, even at doses lower than those used for the present study w15x. We have neither observed the decreased T4 nor the increased rT3. Fasting also results in decreased T3 and increased rT3 w35x, but appears unlikely to be affecting present results, as the patients did not show an increased plasma rT3. The changes reported from samples taken intraoperatively are variable, but most reports show a decrease in T3 and an increase in FT4 w35x without a change in rT3 w1x. T4 may be unchanged, decreased or increased, without any change in TSH. The values from the present patients agree with these reports, except that we have not observed an increased FT4. Changes in thyroid status due to such confounding factors would not explain the differences in the concentration of T4 in the normal brain samples reported by us as compared to those given by others, considering that the circulating T4 levels were normal. As cerebral 5X D-II is influenced by the circulating and cerebral T4 concentrations, and hardly affected by those of T3, present 5X D-II activities reported for the normal brain tissue of the patients are also likely to be representative of the activity in normal subjects. The changes in thyroid status due to these factors could, however, be the cause of the decreased plasma T3 levels of our patients, mostly related to the fact that the samples were obtained intraoperatively. It is therefore possible that present T3 concentrations, and those reported by others, are lower than those of a normal subject. However, results from animal experiments make this unlikely, as brain T3 homeostasis is maintained over a wide range of circulating T3 levels, provided circulating T4 is at least 50% of normal values w13x. 4.2. Tumoral samples The tumoral nature of the sample included as such in the present study was confirmed histologically in all cases. DNA concentrations were also higher in these samples than in the samples of normal surrounding tissue, as would be expected considering that abnormalities in the karyotype or cellular DNA content are a typical finding in a variety of neoplastic diseases w3x, and likely to be the consequence of high proliferative activity. In the present series of samples the protein concentration, and the proteinrDNA ratio were decreased as well, and this might be related to major changes in gene expression. In the tumors, T3 concentrations were the same as in normal paired samples, but decreased when the higher DNA concentration was taken into account to about 70%
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of the mean value found for the normal tissue. The decrease was less marked than that in circulating T3, which decreased to more than 50% of the mean levels for normal subjects. The decrease in the concentration of T3 was not accounted for by changes either in the amount of substrate ŽT4. or in the activity of 5X D-II, which generates T3 from T4 locally, as both were normal. As indicated before for the normal tissue samples, the low circulating T3 is not likely to cause a decrease in the concentration of T3 in the tumor, unless mechanism which maintain tight brain T3 homeostasis are lost, or affected. In the tumor cerebral uptake of T3 might be decreased, or the exit of T3 back into the plasma increased. On the other hand, the opposite might be occurring in the normal brain tissue, in order to preserve normal T3 levels. At present there is very little information regarding such possibilities. The presence of 5D activity has been reported in the human fetal brain w21x and in a few samples of normal adult brain tissue w9x or brain tumors w23x obtained intraoperatively or at autopsy. The present study confirms the presence of 5D activity in about 76% of the tumoral tissue samples, but we have been unable to compare it with that of surrounding normal brain tissue, as only two samples of the latter were available to us Ž226.4 " 14.7 fmolrhrmg protein., and none were included in the study of Mori et al. w23x. The data obtained by us for the tumoral tissue were quite variable, ranging from 0 to 251 fmolrhrmg protein, in agreement with the high variability described by Mori et al. w23x for 20 tumor samples Žrange 0 to 986 fmolrhrmg protein.. This variability is markedly higher than found for the 5X D-II activity in the same tumor samples Žrange 2.3 to 27.6 fmolrhrmg protein.. Both in the present study and in that of Mori et al. w23x practically no 5D activity was found in meningiomas. We found no 5D activity in two adenomas of the anterior pituitary, in agreement with the lack of inner ring activity found in pituitaries. We do not know whether this variability is related to the different embryologic origin of the cells involved in the different types of brain tumors included in the study, or is related to their process of dedifferentiation. The variability of the 5D values reported for normal human fetal cerebral cortex w21x is markedly less. It is therefore difficult to evaluate to what extent the presence of 5D activity in the tumor, which deiodinates T4 and T3 into inactive iodothyronines, could account for the decrease in their T3 content.
Acknowledgements We thank Drs. R. Thoma and H. Rokos ŽHenning, Berlin. for the generous gift of the inner-ring labeled 5-w125 IxT3. We are also in debt with S. Duran, ´ A. Hernandez ´ and M.-J. Presas for invaluable technical assistance. This work was supported by Grant FISS ŽFondo de Investigaciones Sanitarias. 92-0888, Spain.
References w1x H.O. Adami, H. Johansson, L. Thoren, L. Wide, G. Akerstrom, Serum levels of TSH, T3, rT3, T4 and T3-resin uptake in surgical trauma, Acta Endocrinol. 88 Ž1978. 482–489. w2x R. Arem, G.J. Wiener, S.G. Kaplan, H.S. Kim, S. Reichlin, M.M. Kaplan, Reduced tissue thyroid hormone levels in fatal illness, Metabolism 42 Ž1993. 1102–1108. w3x G.U. Auer, U.G. Falkmer, A.D. Zetterberg, Image cytometric nuclear DNA analysis in clinical tumour material, in: J.P.A. Baak ŽEd.., Manual of Quantitative Pathology in Cancer Diagnosis and Prognosis, Springer, Heidelberg, 1991, pp. 211–232. w4x A. Baumgartner, K.J. Graf, I. Kurten, H. Meinhold, The hypothalamic pituitary thyroid axis in psychiatric patients and healthy subjects: Part II. Repeated measurements of thyroxine, free thyroxine, triiodothyronine, free triiodothyronine, and reverse triiodothyronine in patients with major depressive disorder and schizophrenia and healthy subjects, Psychiatry Res. 24 Ž1988. 283–305. w5x J. Bernal, F. Pekonen, Ontogenesis of the nuclear 3,5,3X-triiodothyroxine receptor in the human fetal brain, Endocrinology 114 Ž1984. 677–679. w6x K.A. Burton, A study of the conditions and mechanisms of the diphenilamine reaction for the colorimetric estimation of deoxiribonucleic acid, Biochem. J. 62 Ž1956. 315–323. w7x R. Calvo, M.J. Obregon, ´ C. Ruiz de Ona, ˜ F. Escobar del Rey, G. Morreale de Escobar, Congenital hypothyroidism as studied in rats: X crucial role of maternal thyroxine but not of 3 ,3,5-triiodothyronine in the protection of the fetal brain, J. Clin. Invest. 86 Ž1990. 889–899. w8x R.M. Calvo, G. Morreale de Escobar, F. Escobar del Rey, M.J. Obregon, ´ Maternal non-thyroidal illness and fetal thyroid hormone status, as studied in the streptozotocin-induced diabetes mellitus rat model, Endocrinology 138 Ž1997. 1159–1169. w9x A. Campos-Barros, T. Hoell, A. Musa, S. Sampaolo, G. Stoltenburg, G. Pinna, M. Eravci, H. Meinhold, A. Baumgartner, Phenolic and tyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system, J. Clin. Endocrinol. Metab. 81 Ž1996. 2179–2185. w10x A. Campos-Barros, H. Meinhold, R. Kohler, F. Muller, M. Eravci, A. Baumgartner, The effects of desipramine on thyroid hormone concentrations in rat brain, Naunyn-Schmiedeberg’s Arch. Pharmacol. 351 Ž1995. 469–474. w11x W.H. Crosby, F.W. Furth, A modification of the benzidine method for measurement of hemoglobin in plasma and urine, Blood 11 Ž1956. 380–383. w12x M.B. Dratman, F.L. Crutchfield, J.T. Gordon, A.S. Jennings, Iodothyronine homeostasis in rat brain during hypo- and hyperthyroidism, Am. J. Physiol. 245 Ž1983. ŽEndocrinol. Metab. 8. E185–E193. w13x H.F. Escobar-Morreale, F. Escobar del Rey, M.J. Obregon, G. Morreale de Escobar, Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats, J. Clin. Invest. 96 Ž1995. 2828– 2838. w14x B. Ferreiro, J. Bernal, C.G. Goodyer, C.L. Branchard, Estimation of nuclear thyroid hormone receptor saturation in human fetal brain and lung during early gestation, J. Clin. Endocrinol. Metab. 67 Ž1988. 853–856. w15x A. Gamstedt, G. Jarnerot, B. Kagedal, Dose related effects of betamethasone on iodothyronines and thyroid-binding proteins in serum, Acta Endocrinol. 96 Ž1981. 484–490. w16x B. Gancedo, I. Corpas, G.A. Alonso, M.J. Delgado, G. Morreale de Escobar, B.M. Alonso, Corticotropin-releasing factor stimulates metamorphosis and increases thyroid hormone concentration in prometamorphic Rana perezi larvae, Gen. Comp. Endocrinol. 87 Ž1992. 6–13. w17x R.C. Griggs, Neurologic manifestations of endocrine disfunction, in:
R.M. CalÕo et al.r Brain Research 801 (1998) 150–157
w18x w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x
A.I. Arieff, R.C. Griggs ŽEds.., Metabolic Brain Disfunction in Systemic Disorders, Little Brown, Boston, 1992, pp. 205–215. J.A. Hatterer, J.H. Kocsis, P.E. Stokes, Thyroid function in patients maintained on lithium, Psychiatry Res. 26 Ž1988. 249–257. R.T. Joffe, J.N. Nobrega, S.J. Kish, R. Calvo, L.M. Dixon, J.M. Wilson, G. Morreale de Escobar, Regional thyroid hormone levels in rat brain, Psychoneuroendocrinology 19 Ž1994. 773–777. R.T. Joffe, W. Singer, The effect of tricyclic antidepressants on basal thyroid hormone levels in depressed patients, Pharmacopsychiatry 23 Ž1990. 67–69. X M.G. Karmarkar, D. Prabarkaran, M.M. Godbole, 5 -Monodeiodinase activity in developing human cerebral cortex, Am. J. Clin. Nutrition 57 Ž1993. S291–S294. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. K. Mori, K. Yoshida, T. Kayama, N. Kaise, H. Fukazawa, Y. Kiso, K. Kikuchi, Y. Aizawa, K. Abe, Thyroxine. 5-Deiodinase in human brain tumors, J. Clin. Endocrinol. Metab. 77 Ž1993. 1198–1202. G. Morreale de Escobar, R. Calvo, F. Escobar del Rey, M.J. Obregon, Thyroid hormones in tissues from fetal and adult rats, Endocrinology 134 Ž1994. 2410–2415. G. Morreale de Escobar, R. Calvo, M.J. Obregon, F. Escobar del Rey, Homeostasis of brain T3 in rat fetuses and their mothers: effects of thyroid status and iodine deficiency, Acta Med. Austriaca 1 Ž1992. 110–116. G. Morreale de Escobar, F. Escobar del Rey, A. Ruiz Marcos, Thyroid hormone and the developing brain, in: J.H. Dussault, P. Walker ŽEds.., Congenital Hypothyroidism, Marcel Dekker, New York, 1983, pp. 85–126. G. Morreale de Escobar, R. Pastor, M.J. Obregon, ´ F. Escobar del
w28x
w29x
w30x
w31x w32x
w33x
w34x
w35x
157
Rey, Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, Endocrinology 117 Ž1985. 1890–1901. M.J. Obregon, ´ A. Pascual, G. Morreale de Escobar, F. Escobar del Rey, Pituitary and plasma thyrotropin, thyroxine and triiodothyronine after hyperthyroidism, Endocrinology 104 Ž1979. 1467–1473. C. Ruiz de Ona, ˜ G. Morreale de Escobar, R. Calvo, F. Escobar del Rey, M.J. Obregon, ´ Thyroid hormones and 5X-deiodinase in the rat fetus late in gestation: effects of maternal hypothyroidism, Endocrinology 128 Ž1991. 422–432. C. Ruiz de Ona, ˜ M.J. Obregon, ´ F. Escobar del Rey, G. Morreale de X Escobar, Developmental changes in rat brain 5 -deiodinase and thyroid hormones during the fetal period: the effects of fetal hypothyroidism and maternal thyroid hormones, Pediatr. Res. 24 Ž1988. 588–594. G.W. Snedecor, W.G. Cochran, Statistical Methods, 7th edn., Iowa State Univ. Press, Ames. D. van der Heide, M.P. Ende Visser, T4, T3 and reverse T3 in the plasma of rats during the first 3 months of life, Acta Endocrinol. 93 Ž1980. 448–454. J. van Doorn, F. Roelfsema, D. van der Heide, Concentrations of X thyroxine and 3,5,3 -triiodothyronine at 34 different sites in euthyroid rats as determined by an isotopic equilibrium technique, Endocrinology 117 Ž1985. 1202–1208. T.J. Visser, J.L. Leonard, M.M. Kaplan, P.R. Larsen, Kinetic eviX dence suggesting two mechanisms for iodothyronine 5 -deiodination Ž . in rat cerebral cortex, Proc. Natl. Acad. Sci. 79 1982 5080–5084. L. Wartofski, K.D. Burman, Alterations in thyroid function in patients with systemic illness: the ‘euthyroid sick syndrome’, Endocrine Rev. 3 Ž1982. 164–217.
Research report
Thyroid hormones in human tumoral and normal nervous tissues Rosa Marıa ´ Calvo a
a, )
1
, Jose Marıa ´ Roda b , Marıa ´ Jesus ´ Obregon ´ a , Gabriella Morreale de Escobar
a
Unidad de Endocrinologıa del CSIC and Facultad de Medicina, UAM, Madrid, Spain ´ Molecular, Instituto de InÕestigaciones Biomedicas ´ b SerÕicio de Neurocirugıa, ´ Hospital La Paz, Madrid, Spain Accepted 26 May 1998
Abstract We have studied T4 and T3 concentrations, DNA and protein concentrations and 5X and 5 deiodinases in samples of brain tumors obtained at surgery from 49 patients, and, in most cases, also from surrounding normal tissue. T4 concentrations in normal cortical tissue Ž6.19 " 0.45 ngrg. were lower than in white matter, but the difference disappeared when referred to the DNA content Ž2.26 " 0.27 ngrmg DNA.. No other differences were found between cortical and white matter, or among cortical lobes. T4 in normal tissue was higher than previously reported, mostly from autopsy samples, whereas T3 Ž0.99 " 0.07 ngrg. was similar. 5X D-I activity was negligible as compared to 5X D-II Ž8.11 " 1.09 fmolrhrmg protein.. When expressed in relation to the different DNA contents of normal vs. tumoral tissue, 5X D-II activities were the same for both. 5D activity was highly variable in the tumoral tissue, with negligible activities in meningiomas and pituitary adenomas. When referred to the DNA content, T4 and 5X D-II were the same, but T3 concentrations were lower in the tumor Ž0.24 " 0.03 ngrmg DNA. as compared to normal Ž0.35 " 0.04 ngrmg DNA. tissue samples. Whether or not this decrease of T3 affects the expression of T3-sensitive processes remains to be studied. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Thyroxine; Triiodothyronine; Deiodinase; Human; Brain; Tumor
1. Introduction The thyroid hormones, L-thyroxine ŽT4. and 3,5,3X-triiodo-L-thyronine ŽT3., are not only essential for normal brain maturation, but also for normal brain function throughout life w17,26x. Most of the information on the concentrations of T4 and T3 in adult brain, the activities of the iodothyronine deiodinases involved in outer Ž5X D-I and 5X D-II. and inner ring Ž5D. deiodination, the local generation of T3 from T4 and the regulatory mechanisms maintaining tight homeostatic control of cerebral T3 levels has been obtained in the rat w7,12,13,25x. The possible involvement of thyroid hormones in the response to treatment of patients with severe depression w4,18,20x has renewed the interest in cerebral thyroid hormone levels and metabolism in the adult brain. Although the iodothyronine concentra-
) Corresponding author. Instituto Investigaciones Biomedicas, Arturo ´ Duperier 4, 28029 Madrid, Spain. Fax: q34-1-5854587; E-mail: [email protected] 1 Presented at the 22nd Annual Meeting of the European Thyroid Association, Viena, Austria, 1994.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 7 6 - 9
tions and the deiodinase activities in the developing human cerebral cortex of aborted fetuses have been reported w5,14,21x, there is very little information on the adult human brain because of the obvious difficulties involved. The thyroid hormone concentrations in brain and other tissues obtained from victims of sudden accidental death and from patients who died after fatal illness have been reported; however, the time elapsed between death and autopsy was up to 26 h w2x. More recently, another study w9x has also investigated the iodothyronine levels in nontumoral nervous tissue obtained during surgery or at autopsy Žwith delays of up to 72 h.. Regarding the monodeiodinating enzymes, there is a report on 5D in human brain tumors w23x and another on the biochemical properties of 5X D-II and 5D in normal human central nervous system ŽCNS. as measured in vitro w9x. We have measured the concentrations of T4 and T3, and the activities of 5X D-I, 5X D-II and 5D in samples from cerebral tumors and, whenever ethically possible, from an aliquot of the sample of the presumably normal tissue taken for morphological examination. In addition, we have compared the above parameters among normal cortical lobes and between normal cerebral cortex and white matter, as well as between tumoral and normal tissue.
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2. Materials and methods 2.1. Patients and tissue sampling In the first part of the study, 35 patients have been involved, 15 men and 20 women from the Hospital La Paz, Madrid. Their ages ranged from 15 to 78 years Ž48.4 " 2.7.. One patient was operated on for the removal of a gliosis and the rest for the removal of brain tumors of different types: 10 astrocytomas of all histological grades, eight glioblastomas, four meningiomas, two oligodendrogliomas, two oligoastrocytomas, two metastases and a ganglioglioma, a glioma, a neurinoma, a cordoma, a hamartoma, and an adenoma of the anterior pituitary. The patients were not known to be affected by any thyroid disorder. They were on dexamethasone Ž16 mgrday; 4 mg every 6 h. for 3 to 5 days before surgery. The patients were fasted overnight, and the tissue samples were obtained after 1–2 h of endotracheal anesthesia. Specimens of normal brain tissue, either preferably cerebral cortex or white matter were obtained in lobectomized brains; to minimize the influence of edema, they were taken from the zone most distal to the periphery of the tumor. An aliquot of the ablated tumor, and of the normal tissue were rapidly frozen in liquid nitrogen and kept at y708C until the determinations were carried out. Samples available for the present study weighed from 31 to 880 mg. The amount of tissue was often too small to perform all the analytical determinations. None of the patients had been treated by radiotherapy before the operation. In the second part of the study, tissue samples from another 14 patients were collected. Of these subjects, seven were female and seven were male with ages ranging from 38 to 77 Ž55.3 " 2.9.. The tissue samples were obtained from four meningiomas, two neurinomas, one astrocytoma, four glioblastomas, two pituitary adenomas and one aliquot of normal temporal cortex tissue obtained at surgery for epilepsy. In most cases Ž45 patients. a blood sample was obtained from the forearm during the operation, for the determination of plasma T4, T3, rT3, free T4 and thyrotropin ŽTSH. levels. The above parameters were also assayed in the plasma from 45 healthy volunteers of both sexes and similar ages working at our institute to obtain a normal range in a control population. All the investigations were performed in accordance with the guidelines in the Declaration of Helsinki and approved by the Ethics Committee of the Hospital La Paz, Madrid.
151
as described elsewhere in detail w27x. In brief, the sample is homogenized directly in methanol, and w131 IxT4 and w125 IxT3 are added to each sample as internal tracers for recovery calculations. These tracers are added in amounts small enough to avoid interferences in the final RIAs. Appropriate volumes of chloroform are added to the extract with chloroform:methanol Ž2:1, v:v., twice, followed by backextraction into an aqueous phase, also twice. The aqueous phase from each back-extraction is pooled and further purified on Bio-Rad 1 = 2 resin columns, from which the iodothyronines are eluted with 70% acetic acid. The eluates containing the labeled tracers are pooled, evaporated to dryness and extensively counted for the calculation of individual recoveries. RIA buffer is then added, and the T4 and T3 contents determined by RIAs in triplicate at two dilutions. For the determination of rT3 we used the same procedure as for T4 and T3, but using w125 IxrT3 as recovery tracer. The limits of detection are 2.5 pg T4, 0.7 pg T3 and 1 pg rT3rtube. The molar cross-reactivities for the RIAs and the inter and intraassay variations Žbelow 10%. have been previously described w16,28,29,32x. 2.3. Determination of deiodinase actiÕities 5X D-II activity was assayed in normal and tumoral brain samples, as described w30x. Before each assay w125 IxrT3 or w125 IxT4 were purified by paper electrophoresis to separate the contaminating iodide. 5X D-II activity was assayed incubating 50–200 mg protein in 100 mM potassium phosphate buffer ŽpH s 7.0., 1 mM EDTA with approximately 80,000 cpm of w125 IxrT3, 2 nM rT3, 20 mM DTT, in the presence or absence of 1 mM 2-N-propyl-6-thiouracil ŽPTU. for 1 h at 378C. The total volume was 100 ml. The activity which was not inhibited by PTU was considered as 5X D-II, and the PTU-sensitive activity as 5X D-I. Some samples Ž n s 11. were also assayed using w125 IxT4, 2 nM T4 q 1 mM T3 and 20 mM DTT, as previously reported w30x. The 125 Iodide release was separated by ion-exchange chromatography on Dowex-50W-X2 columns equilibrated in 10% acetic acid. The production of equal amounts of iodide and 3X ,3-T2 was checked in some assays. The protein content was determined by the method of Lowry et al. w22x, after precipitation of the homogenates with 10% TCA to avoid interferences from DTT in the colorimetric reaction. 5D activity was measured as described w8x by incubating 40–50 mg protein in 100 mM potassium phosphate buffer ŽpH s 7.4., 1 mM EDTA with approximately 50,000 cpm of inner-ring labeled 5-w125 IxT3, 25 nM T3, 20 mM DTT and 1 mM PTU for 1 h at 378C.
2.2. Determination of T4, T3 and rT3
2.4. Other determinations
T4 and T3 were determined by highly sensitive and specific radioimmunoassays after extensive extraction and purification of the iodothyronines from tissues or plasma,
The concentration of DNA and protein were also determined in aliquots of the tissue homogenates by the methods of Burton w6x and Lowry et al. w22x, respectively.
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Hemoglobin content was also measured in the tissue samples, as described by Crosby and Furth w11x, to assess the possible contribution of the thyroid hormones contained in blood trapped in the tissue sample. As T4 and T3 were measured in serum, a mean value of 50% was assumed for the hematocrit in order to calculate this possible contribution. Serum TSH was assayed by the Dynotest TSH kit ŽHenning Berlin, Berlin, Germany.. The lower limit of sensitivity is 0.16 mIUrml. Free T4 was measured using the Two-Step Gammacoat Free T4 RIA kit from Clinical Assays ŽBaxter, Cambridge, MA.. 2.5. Drugs and reagents T4, T3 and 3,5-diiodo-thyronine Ž3,5-T2., PTU and were from Sigma ŽSt Louis, MO.. Reverse T3 ŽrT3. and 3X ,3-diiodothyronine Ž3X ,3-T2. were from Henning Berlin, Bio-Rad 1 = 2 resin from Bio-Rad Laboratories ŽRichmond, CA.. High specific activity w125 IxT3, w125 IxT4, w125 IxrT3, 131 w IxT4 Ž3000 mCirmg. were synthesized by us, as described w27x. Inner-ring labeled 5-w125 IxT3 Ž80 mCirmg. was used as substrate for 5-deiodinase. It was provided by Drs. R. Thoma and H. Rokos from Henning Berlin. DL-dithiothreitol
2.6. Statistical analysis Results are shown as mean values " S.E.M. Significance of differences between groups was measured by Student’s t-test when only two groups were compared, and by one-way analysis of variance in the case of three or more groups. All calculations, including correlations, were performed as outlined by Snedecor and Cochran w31x.
3. Results 3.1. Circulating parameters of thyroid hormone status The concentration of T4, T3, rT3, TSH and circulating free T4 were measured in the plasma of these patients, as well as in a group of healthy adults. Total plasma T4 levels were normal whereas total plasma T3 concentrations were decreased when compared to those of healthy human beings, as all of them were below the normal range ŽTable 1.. Plasma rT3 in the patients with brain tumors fell in the Table 1 Mean values Ž"S.E.M.. of T4, T3, rT3, TSH and FT4 in the plasma obtained from patients bearing brain tumors ŽA.; and the normal range of control adults values obtained from 45 healthy volunteers ŽB. T4 Žmgrdl. T3 Žngrdl. rT3 Žngrdl. TSH ŽmIUrml. FT4 Žngrdl. A 7.6"0.4 B 3.1–7.8
58.3"2.7 137–293
21.2"1.8 20–82
1.0"0.2 0.5–2.6
1.7"0.2 1.4–2.8
lower range of normal people. Results of circulating TSH and free T4 were also within the normal range of our control values. 3.2. Normal cerebral tissue 3.2.1. Cerebral cortical tissue Õs. white matter In presumably normal cerebral tissue, we did not find any statistically significant differences between the concentrations of thyroid hormone, deiodinases, DNA and protein, in samples from the cerebral cortex and underlying white matter, except for the concentration of T4 which was higher in white matter when expressed per gram of wet tissue ŽTable 2.. This difference was also found when the data were calculated only with paired tissue samples, namely, those obtained from the same patient. However, it was no longer found when the concentration of T4 was referred to DNA content instead of weight of the sample. 3.2.2. Cortical lobes of the brain We determined the same parameters as above in different areas of the cerebral cortex: frontal, parietal, occipital and temporal lobes of presumably normal tissue. We did not observe any significant change between areas in the concentrations of T4, T3 and DNA and in deiodinase activities. Only the protein concentration was lower in the parietal lobe of the cortex Ždata not shown.. 3.3. Tumoral Õs. normal cerebral tissue Fig. 1 shows changes in concentrations of DNA and protein and in the proteinrDNA ratio in tumoral vs. normal brain paired samples. As may be seen, DNA increased whereas the protein and proteinrDNA ratio decreased in malignant vs. normal cerebral tissue. T4 and T3 levels and 5X D-II activities are presented in Fig. 2. Hemoglobin was measured to assess the possible contribution to the tissue concentration of thyroid hormones which were actually contained in trapped blood. The results shown in Fig. 2 were not affected, because in all cases, except one, the hormone levels in the trapped blood represented less than 1% of the value. T4 concentration was 1.8 times higher in tumoral as compared to normal tissue, but the change disappeared when values were referred to the DNA content. On the contrary, there was no difference in T3 levels when expressed per gram of wet weight of the tissue, but when referred to the DNA content, there was a decrease to about 70% of the normal values. 5X D-II activity showed a pattern similar to that observed for the T4 concentration: high when expressed per gram of weight and the same when referred to DNA content. Fig. 3 shows the concentrations of T4 and T3 and the 5X D-II activity in the different types of brain tumors. The T4 concentration ranged from 45.1 ngrg in one of the
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Table 2 X Concentrations of T4, T3, DNA and protein, as well as type II 5 D activity, in samples obtained from cortical cerebral tissue as compared to underlying white matter
ng T4rg ng T3rg X 5 D-II Žfmolrhrmg protein. mg DNArg mg proteinrg ng T4rmg DNA ng T3rmg DNA X 5 D-IIrmg DNA Žfmolrhrmg DNA.
Cortical tissue
White matter
t statistic
6.19 " 0.45 Ž22. 0.99 " 0.07 Ž26. 8.11 " 1.09 Ž21. 3.0 " 0.2 Ž25. 92.8 " 4.6 Ž22. 2.26 " 0.27 Ž22. 0.39 " 0.05 Ž25. 243 " 35 Ž21.
11.89 " 2.21 Ž7.)) 1.17 " 0.11 Ž6. 9.16 " 1.52 Ž5. 3.8 " 0.6 Ž5. 100.8 " 11.6 Ž8. 2.53 " 0.40 Ž6. 0.51 " 0.17 Ž7. 374 " 120 Ž5.
P - 0.005 n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s., not significant. Values are means" S.E.M. The numbers in parentheses indicate the number of samples involved.
In order to explore the presence of 5X D-I deiodinase, in the first set of brain samples we have determined the activity of the 5X-D enzymes using w125 Ix-rT3 as substrate. We found that 5X D-I activity Žnot shown. was very low both in tumoral and normal tissues, with undetectable values in 50% of the samples. Even when the activity was
astrocytomas to 1.6 ngrg in the hamartoma. The highest concentration of T3 Ž3.5 ngrg. was found in an adenoma of the anterior pituitary whereas two glioblastomas showed the lowest level of T3 Ž0.23 ngrg.. 5X D-II activity ranged from 657 fmolrhrmg protein in the adenoma to 0.64 fmolrhrmg protein in one of the meningiomas.
Fig. 1. DNA and protein concentrations, and proteinrDNA ratio Žmean " S.E.M.. in paired samples from brain tumors and surrounding normal tissue. )Identifies a statistically significant difference Ž p - 0.05..
X
Fig. 2. Concentrations of T4 and T3 Žexpressed per gram of wet weight. and 5 D-II activity in normal and tumoral samples from human brain Ždotted bars.. Darker bars represent the same parameters referred to DNA content. )Identifies a statistically significant difference in tumoral vs. normal brain samples Ž p - 0.05.. Data are means" S.E.M. The numbers in circles are the number of observations.
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detected, in the other 50% of the samples 5X D-I represented less than 10% of the total 5X D activity. In view of the very low PTU-sensitive 5X D activity, further samples were assayed using T4 as the preferred substrate for this assay. Moreover, rT3 was also used in parallel, in order to validate the results of the first set of samples. The enzyme was determined in crude homogenates; no microsomal fractions or kinetic studies could be performed because of the very small amount of tissue available. Results obtained were comparable. Both were correlated positively Ž n s 11; y s 0.353 x q 4.525, r s 0.833; with the 5X D-II activity using T4 in the abscissa and the activity using rT3 in ordinates.. The means obtained were 9.01 " 2.43 and 12.5 " 3.76 fmolrhrmg protein, respectively, when w125 Ix-rT3 or T4 were used. 5D activity was also determined in these second brain tumoral samples and in four of the previous set ŽFig. 4.. Of
Fig. 4. 5D-III activity in brain tumors of different types, as well as in two samples from normal surrounding cortex ŽCx.. The identification of tumoral samples is the same as in Fig. 3.
17 tumors, only 13 contained 5D activity, the mean " S.E.M. being 40.33 " 17.16 fmolrhrmg protein. The tyrosyl ring deiodinase activity was also assayed in two normal cortical samples Ž226.4 " 14.65 fmolrhrmg protein.. Only two tumors Žone astrocytoma and one glioblastoma. showed higher 5D activity than that of the normal tissue samples. Meningiomas Ž n s 5. either showed no or very low inner ring deiodinase activity. Two adenomas of the anterior pituitary exhibited no 5D activity. No clear correlations were found for the concentration of T4, T3 or the deiodinase activities with age, sex, type of tumor or degree of malignancy.
4. Discussion Thyroid hormones play an essential role in brain development and function throughout life. Most evidence has been obtained from studies in animal models, and information in humans is scarce. In our study T4, T3, rT3 and 5X and 5-monoiodinases were determined in tumoral and normal tissue from different zones of the CNS obtained from living patients at surgery. 4.1. Normal cerebral tissue
Fig. 3. Concentrations of T4 and T3 Žexpressed per gram of wet weight. X and 5 D-II activity in different types of brain tumors. The identification of tumors is as follows: As: astrocytoma, Gb: glioblastoma, Me: meningiomas, Mt: metastases, Oa: oligoastrocytoma, Od: oligodendroglioma, Ne: neurinoma, Co: cordoma, Ha: hamartoma, Gl: glioma, Gg: ganglioglioma, Ad: adenoma.
Deiodinase activities were measured in normal brain samples, practically all the phenolic ring deiodinase activity present appears to be 5X D-II. To our knowledge these are the first results of 5X D-II activities in the four lobes of the cortex from human adults, as only data for the temporal lobe have been published previously w9x. 5X D-II activity has been reported in human fetal cortex w21x. Although 5X D-I has been reported in the rat w34x, the activity of 5X D-I activity in our human tissue samples was negligible. This is in agreement with Campos-Barros et al. w9x who did not find a PTU-sensitive 5X-deiodination, also suggesting the absence of 5X D-I activity in the adult human brain. Thyroid hormone concentrations were also determined in normal brain tissue but we did not find differences in
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either T4 or T3 concentrations among the four lobes of the cortex. This is in agreement with a recent report w9x, although thyroid hormones were only studied in the temporal and frontal lobes of individuals at autopsy or surgery. This might suggest that there is not a specific pattern of distribution of the iodothyronine levels in the lobes of the cerebral cortex, however, it should be pointed out that we have grouped the tissue samples according to a rough localization, which may not be representative of the whole lobe. In addition, we cannot exclude from present results that there is no region-specific distribution of T4 and T3 in the rest of the brain. In the rat we have shown higher T4 levels Žin ngrg. in cerebellum and thalamus and high T3 in thalamus, but both hormones are consistently distributed across other brain areas, cortex included w19x. Cortex T3 levels are comparable to those reported for the rat w10,19,33x and also to those reported in human brain samples obtained intraoperatively w9x, from victims of sudden death w2,9x and from human fetuses w21x. Tissue T4 levels are comparable to those found in developing fetal human brain w21x, but are 2.5 to 4.5 times higher than in the rat w10,19,33x. Our T4 values are also higher than that reported in two studies for victims of accidental death but the time elapsed between death and autopsy was up to 26 h w2x, and 72 h w9x, respectively. During this time, some degradation of T4 and T3 might occur as the 5D enzyme is active in tissues obtained postmortem w9x. T4 concentration in normal brain samples obtained at tumor excision or at resection in living patients was also clearly lower than that we report here, but only four samples were studied w9x. The possible cause for the difference among reports in the concentration of T4 has not been clarified. Hemoglobin was measured in the brain samples of our study to determine if trapped blood could account for the higher intracerebral T4 levels; however, the contribution was less than 1%. We have compared the ratio between the brain T4 and the total serum T4 of the present patients and that obtained by us in the adult rat w24x, and it is more similar Ž0.09 and 0.08, respectively. than that obtained for autopsied humans Ž0.035. w2x. We believe that present concentrations of T4 and T3 in normal brain tissue from patients with a brain tumor are likely to be more similar to those of normal people than values obtained at autopsy, but we cannot exclude other possible factors considering that our patients had low circulating T3. Factors which might be affecting the values found by us are the fact that the patients had a brain tumor and that the samples were obtained after a few days on corticosteroids and while the patient was undergoing surgery, involving anesthesia, all of which are not physiological circumstances. Malignancy has been observed to influence thyroid status, with alterations typical of non-thyroidal illness, where the most common findings are a decreased circulating T3 and an increased rT3, a normal T4 and FT4, without a compensatory increase in TSH. Most data were
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obtained from patients with lymphoma, breast and lung cancer, and carcinoma of the colon. We are not aware of studies on patients with brain tumors. In the present series we have not found an increase in rT3, but rather the opposite. Findings in patients with high glucocorticoid levels or after treatment with this type of steroids present both a low T3 and T4, and increased rT3, even at doses lower than those used for the present study w15x. We have neither observed the decreased T4 nor the increased rT3. Fasting also results in decreased T3 and increased rT3 w35x, but appears unlikely to be affecting present results, as the patients did not show an increased plasma rT3. The changes reported from samples taken intraoperatively are variable, but most reports show a decrease in T3 and an increase in FT4 w35x without a change in rT3 w1x. T4 may be unchanged, decreased or increased, without any change in TSH. The values from the present patients agree with these reports, except that we have not observed an increased FT4. Changes in thyroid status due to such confounding factors would not explain the differences in the concentration of T4 in the normal brain samples reported by us as compared to those given by others, considering that the circulating T4 levels were normal. As cerebral 5X D-II is influenced by the circulating and cerebral T4 concentrations, and hardly affected by those of T3, present 5X D-II activities reported for the normal brain tissue of the patients are also likely to be representative of the activity in normal subjects. The changes in thyroid status due to these factors could, however, be the cause of the decreased plasma T3 levels of our patients, mostly related to the fact that the samples were obtained intraoperatively. It is therefore possible that present T3 concentrations, and those reported by others, are lower than those of a normal subject. However, results from animal experiments make this unlikely, as brain T3 homeostasis is maintained over a wide range of circulating T3 levels, provided circulating T4 is at least 50% of normal values w13x. 4.2. Tumoral samples The tumoral nature of the sample included as such in the present study was confirmed histologically in all cases. DNA concentrations were also higher in these samples than in the samples of normal surrounding tissue, as would be expected considering that abnormalities in the karyotype or cellular DNA content are a typical finding in a variety of neoplastic diseases w3x, and likely to be the consequence of high proliferative activity. In the present series of samples the protein concentration, and the proteinrDNA ratio were decreased as well, and this might be related to major changes in gene expression. In the tumors, T3 concentrations were the same as in normal paired samples, but decreased when the higher DNA concentration was taken into account to about 70%
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of the mean value found for the normal tissue. The decrease was less marked than that in circulating T3, which decreased to more than 50% of the mean levels for normal subjects. The decrease in the concentration of T3 was not accounted for by changes either in the amount of substrate ŽT4. or in the activity of 5X D-II, which generates T3 from T4 locally, as both were normal. As indicated before for the normal tissue samples, the low circulating T3 is not likely to cause a decrease in the concentration of T3 in the tumor, unless mechanism which maintain tight brain T3 homeostasis are lost, or affected. In the tumor cerebral uptake of T3 might be decreased, or the exit of T3 back into the plasma increased. On the other hand, the opposite might be occurring in the normal brain tissue, in order to preserve normal T3 levels. At present there is very little information regarding such possibilities. The presence of 5D activity has been reported in the human fetal brain w21x and in a few samples of normal adult brain tissue w9x or brain tumors w23x obtained intraoperatively or at autopsy. The present study confirms the presence of 5D activity in about 76% of the tumoral tissue samples, but we have been unable to compare it with that of surrounding normal brain tissue, as only two samples of the latter were available to us Ž226.4 " 14.7 fmolrhrmg protein., and none were included in the study of Mori et al. w23x. The data obtained by us for the tumoral tissue were quite variable, ranging from 0 to 251 fmolrhrmg protein, in agreement with the high variability described by Mori et al. w23x for 20 tumor samples Žrange 0 to 986 fmolrhrmg protein.. This variability is markedly higher than found for the 5X D-II activity in the same tumor samples Žrange 2.3 to 27.6 fmolrhrmg protein.. Both in the present study and in that of Mori et al. w23x practically no 5D activity was found in meningiomas. We found no 5D activity in two adenomas of the anterior pituitary, in agreement with the lack of inner ring activity found in pituitaries. We do not know whether this variability is related to the different embryologic origin of the cells involved in the different types of brain tumors included in the study, or is related to their process of dedifferentiation. The variability of the 5D values reported for normal human fetal cerebral cortex w21x is markedly less. It is therefore difficult to evaluate to what extent the presence of 5D activity in the tumor, which deiodinates T4 and T3 into inactive iodothyronines, could account for the decrease in their T3 content.
Acknowledgements We thank Drs. R. Thoma and H. Rokos ŽHenning, Berlin. for the generous gift of the inner-ring labeled 5-w125 IxT3. We are also in debt with S. Duran, ´ A. Hernandez ´ and M.-J. Presas for invaluable technical assistance. This work was supported by Grant FISS ŽFondo de Investigaciones Sanitarias. 92-0888, Spain.
References w1x H.O. Adami, H. Johansson, L. Thoren, L. Wide, G. Akerstrom, Serum levels of TSH, T3, rT3, T4 and T3-resin uptake in surgical trauma, Acta Endocrinol. 88 Ž1978. 482–489. w2x R. Arem, G.J. Wiener, S.G. Kaplan, H.S. Kim, S. Reichlin, M.M. Kaplan, Reduced tissue thyroid hormone levels in fatal illness, Metabolism 42 Ž1993. 1102–1108. w3x G.U. Auer, U.G. Falkmer, A.D. Zetterberg, Image cytometric nuclear DNA analysis in clinical tumour material, in: J.P.A. Baak ŽEd.., Manual of Quantitative Pathology in Cancer Diagnosis and Prognosis, Springer, Heidelberg, 1991, pp. 211–232. w4x A. Baumgartner, K.J. Graf, I. Kurten, H. Meinhold, The hypothalamic pituitary thyroid axis in psychiatric patients and healthy subjects: Part II. Repeated measurements of thyroxine, free thyroxine, triiodothyronine, free triiodothyronine, and reverse triiodothyronine in patients with major depressive disorder and schizophrenia and healthy subjects, Psychiatry Res. 24 Ž1988. 283–305. w5x J. Bernal, F. Pekonen, Ontogenesis of the nuclear 3,5,3X-triiodothyroxine receptor in the human fetal brain, Endocrinology 114 Ž1984. 677–679. w6x K.A. Burton, A study of the conditions and mechanisms of the diphenilamine reaction for the colorimetric estimation of deoxiribonucleic acid, Biochem. J. 62 Ž1956. 315–323. w7x R. Calvo, M.J. Obregon, ´ C. Ruiz de Ona, ˜ F. Escobar del Rey, G. Morreale de Escobar, Congenital hypothyroidism as studied in rats: X crucial role of maternal thyroxine but not of 3 ,3,5-triiodothyronine in the protection of the fetal brain, J. Clin. Invest. 86 Ž1990. 889–899. w8x R.M. Calvo, G. Morreale de Escobar, F. Escobar del Rey, M.J. Obregon, ´ Maternal non-thyroidal illness and fetal thyroid hormone status, as studied in the streptozotocin-induced diabetes mellitus rat model, Endocrinology 138 Ž1997. 1159–1169. w9x A. Campos-Barros, T. Hoell, A. Musa, S. Sampaolo, G. Stoltenburg, G. Pinna, M. Eravci, H. Meinhold, A. Baumgartner, Phenolic and tyrosyl ring iodothyronine deiodination and thyroid hormone concentrations in the human central nervous system, J. Clin. Endocrinol. Metab. 81 Ž1996. 2179–2185. w10x A. Campos-Barros, H. Meinhold, R. Kohler, F. Muller, M. Eravci, A. Baumgartner, The effects of desipramine on thyroid hormone concentrations in rat brain, Naunyn-Schmiedeberg’s Arch. Pharmacol. 351 Ž1995. 469–474. w11x W.H. Crosby, F.W. Furth, A modification of the benzidine method for measurement of hemoglobin in plasma and urine, Blood 11 Ž1956. 380–383. w12x M.B. Dratman, F.L. Crutchfield, J.T. Gordon, A.S. Jennings, Iodothyronine homeostasis in rat brain during hypo- and hyperthyroidism, Am. J. Physiol. 245 Ž1983. ŽEndocrinol. Metab. 8. E185–E193. w13x H.F. Escobar-Morreale, F. Escobar del Rey, M.J. Obregon, G. Morreale de Escobar, Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats, J. Clin. Invest. 96 Ž1995. 2828– 2838. w14x B. Ferreiro, J. Bernal, C.G. Goodyer, C.L. Branchard, Estimation of nuclear thyroid hormone receptor saturation in human fetal brain and lung during early gestation, J. Clin. Endocrinol. Metab. 67 Ž1988. 853–856. w15x A. Gamstedt, G. Jarnerot, B. Kagedal, Dose related effects of betamethasone on iodothyronines and thyroid-binding proteins in serum, Acta Endocrinol. 96 Ž1981. 484–490. w16x B. Gancedo, I. Corpas, G.A. Alonso, M.J. Delgado, G. Morreale de Escobar, B.M. Alonso, Corticotropin-releasing factor stimulates metamorphosis and increases thyroid hormone concentration in prometamorphic Rana perezi larvae, Gen. Comp. Endocrinol. 87 Ž1992. 6–13. w17x R.C. Griggs, Neurologic manifestations of endocrine disfunction, in:
R.M. CalÕo et al.r Brain Research 801 (1998) 150–157
w18x w19x
w20x
w21x
w22x
w23x
w24x
w25x
w26x
w27x
A.I. Arieff, R.C. Griggs ŽEds.., Metabolic Brain Disfunction in Systemic Disorders, Little Brown, Boston, 1992, pp. 205–215. J.A. Hatterer, J.H. Kocsis, P.E. Stokes, Thyroid function in patients maintained on lithium, Psychiatry Res. 26 Ž1988. 249–257. R.T. Joffe, J.N. Nobrega, S.J. Kish, R. Calvo, L.M. Dixon, J.M. Wilson, G. Morreale de Escobar, Regional thyroid hormone levels in rat brain, Psychoneuroendocrinology 19 Ž1994. 773–777. R.T. Joffe, W. Singer, The effect of tricyclic antidepressants on basal thyroid hormone levels in depressed patients, Pharmacopsychiatry 23 Ž1990. 67–69. X M.G. Karmarkar, D. Prabarkaran, M.M. Godbole, 5 -Monodeiodinase activity in developing human cerebral cortex, Am. J. Clin. Nutrition 57 Ž1993. S291–S294. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. K. Mori, K. Yoshida, T. Kayama, N. Kaise, H. Fukazawa, Y. Kiso, K. Kikuchi, Y. Aizawa, K. Abe, Thyroxine. 5-Deiodinase in human brain tumors, J. Clin. Endocrinol. Metab. 77 Ž1993. 1198–1202. G. Morreale de Escobar, R. Calvo, F. Escobar del Rey, M.J. Obregon, Thyroid hormones in tissues from fetal and adult rats, Endocrinology 134 Ž1994. 2410–2415. G. Morreale de Escobar, R. Calvo, M.J. Obregon, F. Escobar del Rey, Homeostasis of brain T3 in rat fetuses and their mothers: effects of thyroid status and iodine deficiency, Acta Med. Austriaca 1 Ž1992. 110–116. G. Morreale de Escobar, F. Escobar del Rey, A. Ruiz Marcos, Thyroid hormone and the developing brain, in: J.H. Dussault, P. Walker ŽEds.., Congenital Hypothyroidism, Marcel Dekker, New York, 1983, pp. 85–126. G. Morreale de Escobar, R. Pastor, M.J. Obregon, ´ F. Escobar del
w28x
w29x
w30x
w31x w32x
w33x
w34x
w35x
157
Rey, Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, Endocrinology 117 Ž1985. 1890–1901. M.J. Obregon, ´ A. Pascual, G. Morreale de Escobar, F. Escobar del Rey, Pituitary and plasma thyrotropin, thyroxine and triiodothyronine after hyperthyroidism, Endocrinology 104 Ž1979. 1467–1473. C. Ruiz de Ona, ˜ G. Morreale de Escobar, R. Calvo, F. Escobar del Rey, M.J. Obregon, ´ Thyroid hormones and 5X-deiodinase in the rat fetus late in gestation: effects of maternal hypothyroidism, Endocrinology 128 Ž1991. 422–432. C. Ruiz de Ona, ˜ M.J. Obregon, ´ F. Escobar del Rey, G. Morreale de X Escobar, Developmental changes in rat brain 5 -deiodinase and thyroid hormones during the fetal period: the effects of fetal hypothyroidism and maternal thyroid hormones, Pediatr. Res. 24 Ž1988. 588–594. G.W. Snedecor, W.G. Cochran, Statistical Methods, 7th edn., Iowa State Univ. Press, Ames. D. van der Heide, M.P. Ende Visser, T4, T3 and reverse T3 in the plasma of rats during the first 3 months of life, Acta Endocrinol. 93 Ž1980. 448–454. J. van Doorn, F. Roelfsema, D. van der Heide, Concentrations of X thyroxine and 3,5,3 -triiodothyronine at 34 different sites in euthyroid rats as determined by an isotopic equilibrium technique, Endocrinology 117 Ž1985. 1202–1208. T.J. Visser, J.L. Leonard, M.M. Kaplan, P.R. Larsen, Kinetic eviX dence suggesting two mechanisms for iodothyronine 5 -deiodination Ž . in rat cerebral cortex, Proc. Natl. Acad. Sci. 79 1982 5080–5084. L. Wartofski, K.D. Burman, Alterations in thyroid function in patients with systemic illness: the ‘euthyroid sick syndrome’, Endocrine Rev. 3 Ž1982. 164–217.