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Human and Animal Physiology, Agricultural University, Wageningen, The Netherlands; Henning Berlin (H.R.), Berlin; and Klinische Forschergruppe, Medizinische Poliklinik, Universität Würzburg (J.K.), Wurzburg, Germany; and Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas (G.M.d.E.), Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Ir. J. P. Schröder-van der Elst, Department of Human and Animal Physiology, Agricultural University, Haarweg 10, 6709 PJ Wageningen, The Netherlands. E-mail: Janny.vanderElst{at}ALG.FMD.WAU.NL
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EMD 21388 in vitro inhibits 5'-deiodinase type I in rat liver microsomes (9). In vivo studies with long term treatment of rats with EMD 21388 iv (20 µmol/kg BW·day) showed that this synthetic flavonoid interferes with thyroid hormone secretion, turnover, and metabolism in several tissues in different ways (10, 11, 12, 13). From the double isotope equilibrium study (10), it was clear that in all tissues investigated T4 concentrations decreased. In those tissues that express the 5'-iodothyronine deiodinase isoforms, liver, pituitary, testis, thymus, brown adipose tissue, brain, cerebellum, and hypothalamus, the amounts of T3 locally produced from T4 were decreased. The ratio of [125I]T3/[125I]T4 was unchanged. It was not known whether these effects of EMD 21388 were due to inhibition of deiodinase isoforms or to a shortage of the amount of substrate, T4. As there were tissue-specific changes that differed from the effects expected from work in vitro, especially regarding the activity of the different 5'-deiodinase isoforms, it appeared important to assess whether the flavonoid enters into all organs and, if so, whether it does so similarly in all tissues.
To investigate this phenomenon we studied the distribution of EMD 49209, a congener of EMD 21388 in which bromide is replaced by iodide substituents on the phenolic ring. These iodide atoms were radiolabeled by an exchange labeling reaction with 125I. We used this [125I]radioactive flavonoid together with [131I]T4 to address the question of the changes in the different T4 distribution pools in the intact animal during long term treatment with the flavonoid. We compared their tissue distributions for up to 6 h after a bolus injection in rats pretreated with EMD 21388 iv for 14 days and in rats receiving the vehicle without EMD 21388 pretreatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of radiolabeled compounds
EMD 49209 (10 µg/10 µl dimethylsulfoxide) was diluted with
40 µl phosphate buffer (0.25 M; pH 6.5). To 30 µl
phosphate buffer (0.05 M; pH 7.4) were added chloramine-T
(10 µg/10 µl H2O) and 7.5 µl Na125I (375
µCi); the mixture was vortexed for 2 min at room temperature. EMD
49209 was added immediately and vortexed for 1 min at room temperature;
the radiolabeling was stopped by the addition of 20 µg sodium
bisulfite in 10 µl H2O, followed by 800 µl
H2O. This mixture was purified by HPLC [Waters Associates,
Milford, MA; with UV and radioactivity detectors; Eurosfer-100C10, 5
µm, JF 17 column; flow rate, 1 ml/min; gradient elutions A (5%
acetonitril and 0.1% trifluoroacetic acid) and B (95% acetonitril and
0.1% trifluoroacetic acid) with gradient program 0–30% B in 1.3 min,
30–90% B in 1.3–30 min, 90–100% B in 30–31 min]. EMD 49209 and
its 125I-labeled derivate eluted at 20.8 min; a compound
containing an additional iodine substituent eluted at 23.6 min. The
respective fractions were collected and appropriately diluted. The
specific activity of [125I]EMD 49209 was about 20
µCi/µg with a yield of about 53%.
[131I]T4 was prepared freshly in our laboratory (15). The purity of the radioactivity was assessed by HPLC just before use. 125I or percent 131I was less than 0.1%, and no other labeled metabolites could be detected.
Experiments
On day 14 the rats received a bolus iv injection of 400 µl
[125I]EMD 49209 (3 µCi) and
[131I]T4 (5 µCi) in saline containing 5%
normal rat serum. This bolus injection of [125I]EMD 49209
and [131I]T4 was given 1 h after the
last iv injection of EMD 21388 (Exp B) or vehicle (Exp A); 0.25, 0.5,
1, 2, 3, 4, and 6 h after the injection the rats were bled and
perfused with saline. Tissues were removed, weighed, and counted. To
investigate whether [125I]EMD 49209 is metabolized,
plasma and tissues were extracted and subjected to HPLC chromatography
following the same extraction procedure and HPLC protocol as those used
for the separation of iodothyronines (16). In all tissues analyzed,
[125I]EMD 49209 appeared in the same retention time. With
the exception of free 125I, no other metabolites were
detected. The extraction efficiency for the 125I and
131I activities from all tissues was greater than 95%.
Plasma [125I]EMD 49209 and [131I]T4 disappearance rates and tissue contents of [125I]EMD 49209 and [131I]T4 were determined. The percent doses in tissues were corrected for trapped plasma (17). The intestines of the rats were divided into five segments: three equal lengths of small intestine, cecum, and colon; feces and urine were collected, and for all of these fractions, the percent doses of 125I and 131I were determined.
In total, 30 different organs were dissected and processed. For the sake of clarity, only those organs that contributed substantially to the changes in the distribution of the radioactivity are shown, the other organs are taken together and are shown as "rest." The percent doses in total blood, muscle, fat, skeleton, and skin were calculated according to the relative contribution to body weight (18). The total contents of radioactivity in the intestinal segments and feces were also calculated.
After decay of the 131I initially present in the plasma samples, the concentrations of T4 and T3 were assessed by rat RIA, using 131I-labeled T4 and T3, respectively, as tracers (19). Free T4 levels were measured by ultrafiltration using Microcon 10 (Amicon, Danvers, MA) filters and freshly labeled [131I]T4 (20). Plasma TSH was measured by the specific RIA developed for the rat by the NIDDK (NIH, Bethesda, MD). RP-2 was used as a standard.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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shows the disappearance curves of
[125I]EMD 49209 from plasma. Fifteen minutes after
the bolus injection, [125I]EMD 49209 appeared in tissues
(Table 2A). Peak values were reached between 15–30 min
(Table 2A). The percent dose of [125I]EMD 49209 decreased
in tissues with time. After 6 h, 45% of the dose of
[125I]EMD 49209 was found in the intestines (Table 2A).
In many tissues, such as brain, cerebellum, hypothalamus, and medulla,
the percent dose of 125I radioactivity is very low, ranging
from 0.0001–0.070% of the dose (Table 3). After HPLC
analysis, this activity appears to be iodide. In none of these tissues
could [125I]EMD 49209 be detected. The calculated
tissue/plasma ratios of the individual tissues were more or less
constant during the 6 h, indicating that there was no accumulation
for [125I]EMD 49209 (data not shown).
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shows the disappearance of [131I]T4
from plasma. [131I]T4 disappeared rapidly,
within the first 5–10 min. The uptake of
[131I]T4 by tissues was rapid, especially
that by liver (Table 2B). The percent dose of
[131I]T4 in tissues decreased with time
(Table 2B). In the intestines, about 22% was found after 6 h
(Table 2B).
Comparison between [125I]EMD 49209 and
[131I]T4
distribution. Clear differences in kinetic behavior were evident
(Fig. 1C). [131I]T4 disappeared more
rapidly from plasma, within the first 5–10 min. At the start, the
percent dose of [125I]EMD 49209 was higher than that of
[131I]T4, and then disappeared at a slower
rate than T4, but after 5–6 h, they were at the same
level.
The uptake of [125I]EMD 49209 in tissues was much less
and slower than that of T4. There was a striking difference
in uptake in liver (Table 2, A and B). With time, there was a small
increase in the uptake of [125I]EMD 49209 in muscle and
skin, comparable with that of [131I]T4.
After 6 h the greatest differences were found between the contents of [125I]EMD 49209 and [131I]T4 in liver and intestines.
Exp B: pretreatment of rats with EMD 21388
Distribution of [125I]EMD 49209. The
disappearance of [125I]EMD 49209 from plasma was
greatly enhanced by pretreatment with EMD 21388 (Fig. 1A). EMD 21388
treatment resulted in an increase in the excretion of
[125I]EMD 49209 into the intestines as well as into the
urine (Table 2C). At the first time point (0.25 h), 38% of the
[125I]EMD 49209 was already present in the intestines,
mainly in the first part of the jejunum (Fig. 2, I, II, and III). After
6 h, most of the [125I]EMD 49209 was found in the
intestines (54%) and urine (31%; Table 2C). The radioactivity in the
urine consisted mainly of iodide, indicating that this amount of
[125I]EMD 49209 was metabolized or at least deiodinated.
The percent dose of [125I]EMD 49209 in some organs was
hardly detectable; after 6 h, no [125I]EMD 49209
could be detected in brain, cerebellum, medulla, or hypothalamus. Table 2C shows the percent dose of [125I]EMD 49209 at the
different time points.
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). After 15 min, the uptake of
T4 by liver was very high (Table 2D). After 6 h, a
large part of the T4 was still present in the liver and the
various organs; the total excretion was 40% (intestines, 24%; urine,
16%). After pretreatment with EMD 21388,
[131I]T4 distribution did not change
markedly; there was less T4 in the blood and more in the
organs, and only a slight increase in excretion (Table 2D).
Comparison between [125I]EMD 49209 and
[131I]T4
distribution. Figure 1D shows that pretreatment with EMD 21388
abolished the difference in plasma disappearance of
[125I]EMD 49209 and
[131I]T4 present during vehicle treatment.
After 15 min, the uptake of [131I]T4 by liver
was nearly 10 times higher than that of [125I]EMD 49209
(Table 2, C and D). Figure 2 shows the presence of
[125I] (Fig. 2A) and [131I] (Fig. 2B)
activities in gut fractions with time. After 6 h, approximately
40% of the 125I dose was found in intestinal contents and
feces, whereas only 25% of 131I was present. EMD 21388
treatment caused a shift in the appearance time of 125I in
intestinal fractions. At the first time point, 38% of the dose was
already present in the intestines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two flavonoids used in this study were developed by molecular drug design, directed toward the inhibition of the hepatic 5'-iodothyronine deiodinase. Both fulfill the criteria necessary for the optimal inhibitory potency (4). The only difference between these flavonoids is the two bromide atoms (EMD 21388) vs. the two iodide atoms (EMD 49209) in the 3'- and 5'-positions. This makes it possible that EMD 49209 can be deiodinated by deiodinases; debromination of EMD 21388 is less likely. That deiodination occurs can be seen from the free 125I excreted in urine. This influences the kinetic behavior, which means that the disappearance rate of EMD 49209 is faster than that of EMD 21388; in the vehicle-treated rats, it is about 10% (Exp A), and in EMD 21388-pretreated rats, it is about 20% (Exp B).
From our study it is clear that the synthetic flavonoid, [125I]EMD 49209, disappears very quickly from plasma and enters tissues. However, the percent dose of the flavonoid in the organs is never high and decreases with time. It can be concluded from the rapid uptake of both compounds by the liver that this organ functions as an essential part of the fast pool. However, there is a discrepancy between T4 and the flavonoid. T4 content as a percentage of the dose in the liver is much higher than that of the flavonoid (with a high excretion into the intestinal lumen). This means that in liver, many binding sites exist for T4, but not for the flavonoid.
It appears that EMD 49209 does not bind to intracellular T4 binding sites; there is a rapid transfer from plasma via the liver into the intestines. Also, EMD 21388 does not bind to nuclear thyroid hormone receptor (Bernal, J., personal communication) and does not activate the human 5'deiodinase thyroid hormone-responsive element in LLC-PK1 kidney cell line (Jacoks, T., personal communication). It is possible that without binding to the thyroid hormone-binding sites, there is no retention in tissues. Although these synthetic flavonoids were developed by molecular drug design as T4 analogs (1, 2, 3, 4), the in vivo kinetic behavior of [125I]EMD 49209 is different from that of [131I]T4. This could be explained from the fact that EMD 21388 and EMD 49209 bind only to TTR and not to other plasma proteins, as is the case for T4 (8, 21). This also can explain the differences in the behavior of [125I]EMD 49209 between vehicle-treated and pretreated rats; in vehicle-treated rats, the tracer amount of [125I]EMD 49209 will be totally bound to TTR, and its disappearance will be closely related not only to the binding to TTR, but also to the half-life of TTR. In the pretreated animals, the TTR is completely occupied by the EMD 21388, and the larger part of this flavonoid as well as [125I]EMD 49209 will exist in plasma in the free form. It is surprising that the plasma disappearance curves of EMD and T4 are very similar, whereas their tissue distributions are essentially different.
The higher amount of flavonoid present in the liver will induce hepatic enzymes, i.e. such as those involved in glucuronidation. This would lead to an increased metabolism not only of the flavonoid itself, but also that of T4. It has been shown that the most substantial metabolic pathway of natural flavonoids in mammals is conjugation with glucuronic acid or sulfate (22, 23). The conjugates are excreted in bile and urine. The material excreted by the bile can be hydrolyzed in the intestines by the microflora; the liberated flavonoids may then be reabsorbed. We assume that this is also the case for our synthetic flavonoids.
From our results it is clear that the flavonoid does not cross the blood-brain barrier; therefore, in our view this flavonoid cannot be held directly responsible for the inhibition of 5'-deiodinase type II in brain, cerebellum, hypothalamus, and medulla. Thus, the decrease in T3 locally produced in those tissues should be due to the lowered amount of substrate (T4) (10). The access of the flavonoid to the brain appears completely different from that of the peripheral tissues, indicating that T4 conformational homology is not sufficient to gain access to the brain via the brain-blood barrier or the blood-choriod plexus-cerebrospinal fluid barrier.
Despite the high amount of EMD 21388 in the liver [calculated as: 0.305 (% dose [125I]EMD 49209/g) x 1 µmol (100% dose) = 3.05 nmol/g] compared to that of T4 (0.051 nmol/g) (24), there is no indication that inhibition of 5'-deiodinase type I occurs, as deduced from the unchanged Lc[125I]T3/[125I]T4 ratio in the liver (10). It seems likely that the flavonoids do not reach the deiodinase compartment in tissues despite high transient concentrations of reversible competitive binding of the flavonoid to deiodinases. This is in contrast to other known inhibitors, such as PTU and iopanoic acid, that bind irreversibly to the active site of the enzyme.
Acute and short term treatments with EMD 21388 demonstrate the effects on the displacement of T4 from TTR, transiently increasing free T4 and decreasing TSH (25, 26, 27, 28), whereas long term treatment results in changes in the metabolic pathway of thyroid hormones that cannot only be accounted for by decreased binding of T4 by TTR. Therefore, other mechanisms that have not yet been identified are likely to be responsible (10). The most pronounced change is the strong increase in intestinal excretion and the increased metabolism by the deiodinative pathway, as concluded from the increase in radioactivity in urine.
The differences between in vivo and in vitro effects of the flavonoids can be attributed to the presence of plasma binding proteins in the in vivo situation and their absence in the in vitro incubation system, leading to increased cellular flavonoid concentrations in the in vitro situation. The properties of the flavonoids make them valuable tools in studies of intracellular thyroid hormone regulation in vitro, but also in vivo by influencing T4 production, metabolism, and excretion.
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Acknowledgments |
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Footnotes |
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Received July 23, 1996.
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References |
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