Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

PROSTAGLANDINS

TIIYROIDAUTOREGLJLATION. INIIIBITORYEFFECTSOF JODINATEDDERIVATIVES OF ARACHIKQNIC ACID ON IODINE METABOLISM G.D.Chazenbalk,R.M.Valsecchi,I,.Krawiec,~~.Burton,C;.J.,luvenal,E.Monteagudo,H.A.Chester,M.A.Pisarev Division Bioquimica Nuclear,Gerencia de Radiois&opos,Comisi& Nacional de Energia At6mica;Depto. Qufmica Or&nica,Facultad de Ciencias Exactas y Naturales ,UBA;Buenos Aires ,Argentina ABSTRACT Thyroid autoregulation has been linked to an organi fied iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined.‘Ihe following pure synthetic compounds were prepared:1)14-iodo-lS-hydroxy-5,8,11-eicosatrienoic acid(I-OH-A);Z)its omega lactone(IL-w);3)5-hydroxy-6-iodo-8,ll ,14eicosatrienoic acid delta lactone(IL-$,).Their action on iodine metabol.ism was studied.Iodine uptake was measured in calf thyroid slices. At lo-4M I-OH-A caused a 64% decrease in the T/M ratio while Il.-w inhibited it. by 36% and IL-6 was without effect.At lo- 5M the inhibition was 44% for I-OH-A and 19% for IL-w,while T3 was without action. A possible isotopic dilution effect was excluded,and no change in iodine efflux was observed.The inhibition by 1-011-A of iodide uptake was observ d after only 15 min preincubation.This compound also decreased 12z I accumulation in rats. In calf thyroid slices,I-ai-A at 10s4M,inhibited PB1251 formation by 80%,IL-v by 62% ‘and IL-6 by 37%.T3 and arachidonic acid were without action.I-OH-A also caused a dose-dependent inhibition of TSHstimulated iodide organification. The present results demonstrate,for the first time,that iodinated derivatives of arachidonic acid inhibit thyroid function and mimic the effect of iodide on thyroid autoregulation. INTRODUCTION Excess iodide inhibits different thyroid parameters ,such as iodide uptake and organification(1) ,hormone secretion(2,3) .protein(4) and RNA biosynthesis(S) ,cyclic AMP fonnation(6-8) and thyroid growth(g). Most of these effects are mediated by an intracellular organic iodocompound,but efforts in order to identify it have been tmsuccessful. Thyroid hormones have been proposed to be such intennediates,but their role in thyroid autoregulation is still controversiif(see review,lO).Iodolipids constitute another type of organic iodocompounds synthetized within the gland.We have recently shown that iodinated free fatty acids and neutral lipids constitute the main proportion of thyroid iodolipids (11) .Boeynaems and Hubbard( 12) observed that the rat thyroid releases 5-hydroxy-G-iodo-8,ll ,14-eicosatrienoic acid

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delta lactone(IL-$)to the incubation mediwn.Besides ,the formation of iodinated derivatives of arachidonic acid can be catalyzed by different peroxidases(l3,14). A semi-purified iodinated derivative of arnchidonic acid,ohtaincJ in our laboratory,mimicked the in vitro inhibitory effect of iodide on calf slices(l5) and purified I-OH-A inhibits iodide organification by decreasing the availabili.ty of ff202(16) .In the present report we have further explored the effects of different purified derivatives of arachidonic acid on thyroid iodide metabolism,under basal and stimulated conditions.Our results show that some of these iodinated compounds inhibit thyroid function,and support the hypothesis that they might play a role in thyroid autoregulation. MATERIALS l-In Vitro studies:a)iodide uptake: Calf thyroid was obtained at a slaughterhouse and transported to the laboratory in cold saline.The glands were carefully dissected and slices were obtained with a Stadie-Riggs microtome.Four slices of around 50 mg each(wet weight) were preincubated at 37 C in 4 ml of Krebs-Ringer-phosphate buffer(KRP) pH 7.4,8 mMglucose,during 20-30 min,in order to allow thw release of protein from the slices.Tfiey were then transfered to fresh buffer containing 3 mu methylmercaptoimidazole(MW) and tfre test substances.After 60 min a tracer dose (usually 2 @/ml) of 1251 (NEN,17.4 Ci/mg) was added and the incubations were continued for another 60 min.In some experiments the slices were preincubated i KRP,3 I&I WI during variable periods of time in the presence of lo- PM I-Off-A.They were then transfered to fresh KRP containing 3 mM!+I1 and a tracer dose of radioiodine,and incubated for 60 min.The slices were then extensively washed in cold buffer, blotted on filter paper and weighed.The radioactivity of each slice and of an aliquot of the medium was measured.The T/M ratio was calculated as follows: T/M = c m/m tissue -+i7&GG In another series of studies the slices the T/M ratio was determined by incubating the slices in 40 ml of buffer,the results being essentially the same. An isotope dilution effect was studied as follows:slices were preincubated in 3 n&lMMI,KRPbuffer during 60 min with varying amounts of I-OH-A and KI in order to achieve a total iodideconcentration of lo-4M.At the end of this period the slices were extensively washed i\rith saline or buffer and trasnfered to fresh KRP containing 3 r&l WI1 and radioiodine.The T/M ratio was determined after 60 min. b)iodide efflux: In each experiment 10 slices were preincubated during 30 min in KRF buffer at 37 C.They were then transfered to fresh KRP buffer containing 10-6M KI,3 mMPI% and 10 PCi of 125I.After 60 nun the slices were thoroughly washed and placed individually in vials containing 8 ml of KRP buffer,3 mMMYI + the iodocompounds at lo-SM.Aliquots of the medium were obtained at d?;fferent times(S-90 min) .Calculations

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were performed

according

to Corda and Kohn(l7),applying

Kn = (ln ntl

I In nt2)

the formula:

x 100

t2 tl . where Kn is the rate coefficient,and n 1s the percentage of radioactivity remaining at time t. c) Iodide organification: Slices were prepared as described above,but F&l1was omitted from the buffers.Preincubations were performed during 30 min.The slices were transfered to fresh buffer containg the test substances and after 60 min they were pulse-labeled for another 60 min.At the end of this period the slices were washed with saline,blotted on filter paper and homogenized in 3 mMbMI.Total radioactivity was measured and the homogenate was treated with 20% TCA,followed by two washings of the 1,500 rpm precipitate during 20 min,with cold 10% TCA.The radioactivity in the precipitate was counted and calculated as a percentage of the total. 2-In Vivo studies Female Wistar rats,100 g body weight,were injected ip with 5 klg/ day T3 or I-OII-A,or with 1.25 pg/day KI,during 5 days.The dose of KI administered corresponds to that which would arise from total dehalogenation of I-OH-A.Together with the last injection a tracer dose of 25 pCi of 1251 was administered via ip and the rats were sacrificed 2 hr later.Blood samples were obtained from each animal and serum was separated by centrifugation at 2,500 rpm during 20-30 min at 4 C. The thyroid gland was carefully dissected and weighed.The radioactivity of each gland and of 1 ml of serum was measured,and the T/S ratio was calculated: T/S= c m/ th roid * Since the animals were not pretreated with antit$roid drugs these values correspond to the sum of uptake and organification(iodide accumulation). 3-Preparation of the iodinated derivatives of arachidonic acid Iodinated derivatives of arachidonic acid were prepared.IL-6was obtained by treatment of arachidonic acid with iodide-potassium iodide in aqueous bicarbonate( 18) . I-OH-A and the corresponding omega lactone(IL-W)were prepared from arachidonic acid by the method of Bartlett and Myerson(l9) with slight modifications.All compounds were purified by co unm cllr atography on silicagel or reverse phase fIPLC,and had ifI- and yYC-NMR spectrae in accordance with their structures. All reagents were analytical grade.liovine TSII was obtained cram the National Pituitary Agency(USA) .Statistical analysis was performed according to Dunnet’s test. RESULTS Table 1 shows the comparative effects of the diiferent jodinated derivatives of arachidonic acid on 1251 uptake by calf thyroid slices.The T/M values sflowed some degree of variation in the control

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slices from different experiments.This can be attributed to differen ces in thyroid activity of the animals,probably related different iodide ingestion ,thyroid iodine content or seasonal variations in gland function(l6) .At lo-4M I-OII-A was the most potent(64% inhibition) Comparative E-t. A

action

of several

Treatment Control I-(‘XI-A 10-4M

Table 1 iodocompounds

T/M

P<

on iodide % Inhibition

3.4+0.4

1.470.2

0.01

64

1.9FO.l 2.6TO.2 -

0.01

n.s.

44 24

Control IL- W 10-4M 10-5M

5.2+0.4 3.370.2 4.170.3 -

0.05 n.s.

36 19

C

Control IL- b 10-4M

8.4+0.9 8.7 i 0.4

I1.S.

0

D

Control lo-5M T3

5.4+0.2 4.5yO.6

n.s.

17

lo-5M 10_6M B

Each value

is

the average

uptake

of four

slices

+SEM

when compared to IL-u, (36% inhibition) and to IL-6 (no inhibition). The difference in the biologic potency among these compounds was further confirmed when the dose-response relationship was examined.As can be seen,I-011-A produced a 44% decrease in the T/M ratio at lo-5M,while at lo-6M a 248,non significative,inhibition was observed.When the action of IL-W was examined it caused a non significant 19% inhibition at lo-5M,while no action was observed at 10-6~~~3 at 10s5M had a non significant effect. In order to exclude an isotope dilution effect the following experiments were performed:slices were preincubated in KRP buffer containing 3 fl WI during 60 min in the presence of variable concentrations of KI and 1-011-A enough to make a total iodide concentration of 10_4M, as i.ndicated in Table 2.After this period the slices were washed,placed in fresh KRP buffer,3 mMWI and a tracer dose of radioiodine,and incubated for another 60 min..& can be seen,KI at 10-4M caused a 33% decrease in the T/M values,while I-Of-I-A at 10T4M decreased it by 87%.A dose-response relationship was found,which can only by atributed to I-al-A. In the previous studies the slices had been preincubated with the different test substances for 60 min,followed by a pulse of 1251 of 60 min.In order to learn about the time course of 1-011-A action,the slices were preincubated with lo-5M for different periods.As shown

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Table 2 Influenceof combinationsof I-(X1-A and KI on the T/M ratio Treatment

T/M

Control

26.2+3.7

KI lo-4M

8 Inhibit.

% Inhibit.-Dilution

17.5L2.7 -

33

0

KI 0.75 10-4M + I-OH-A 0.25 10-4M

7.2+0.7*

73

48

KI 0.5 10-4M + 1-W-A 0.5 10-4M

4.7+0.8* -

82

05

2.3+0.2* 3.4+0.2* -

91

83

87

87

KI 0.25 10-4M + I-OH-A 0.75 lo-4M 1-011-A10-4M

Each value is the average of 4 slices+SCM,except for controls(n=12). *:p
Figure 1 Influenceof differentpreincubationtimes on I-oil-A inhibitionof iodide uptake.Sliceswere treatedwith lo-SF1I-MI-A for the indicated times and then T/M ratioswere determined.Each value is the average of 4 slices+SEM.*:p
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in Figure 1 ,after 15 min of preincubation,a 29% decrease in the T/M ratio was found,while preincubations for 30 and 60 min caused inhibitions of 40 and 54 %,respectively. The decrease in the T/M ratio observed can be explained by either a decrease in iodide uptake or an increase in its efflux.When the action of I-OII-A and of IL-won 1251 efflux from prelabeled slices was examined no significant change in iodide efflux rate constant was observed(Figure 2).

x

.% .z z ," a c? F 'E 0 E B

100 90 60 70 60 50 LO 30

20

lob-------_ 15 30

I

45

Ml

75

90

0

15

I

I

I

I

I

30

45

60

75

90

min

Figure 2 Influence on 1251 efflux.Sli.ces were prelabeled,placed in fresh buffer and i.odide efflux was measured as described.Each value is the average of 5-6 slices+SEM. o----ocontrol;o---oIL(A) or I-Oll~A(B) at lo-5M. The reversibility of the inhibitory effect on iodide uptake was also explored.Slices were preincubated for 60 min + the iodocompoimds,washed and placed in fresh buffer for 0 ,45 and 90 iin.T/M ratio was determined for 60 min.As shown in Table 3 a partial recovery was observed after 45 min,with total restoration after 90 min. The action of 1-W-A was also studied in vivo.When rats were treated for 5 days with 5 pg/day of either 1-011-A or T3,T/S ratio decreased by 62 and 82%,respectively.KI(l.Z5 pg/day)decreased it by 27%(tahle 4). This dose of KI corres onds to the amount that would originate from total dehalogenation of I-OII-A. When 1251 organification was analyzed again I-OH-A was the most potent inhibitor(808 i.nhibition at 10-4M)while IL-UJ and IL-$ decreased PBI by 62 and 37%,respectively.AA and T3 were without effect&&able 5) .1-011-A caused a dose-related inhibition(72% inhibition at lo_ ;40% at 10m5M; Table 6).TSII caused a stimulation of PBI formation,and simultaneous addition of 1-011-A decreased significantly the hormone action(Table 6).

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Reversibility

Table 3 of the inhibition of iodide T/M ratios

Pretreatment

0

after

uptake

the indicated 45

9 22

50.8+1.5

1-011-t\ 10-5M

41.020.7

20

52.8+2.1 48.2+1.1 _

IL-w

33.5+1 - .o

35

40.7+2.7 _

10-5M

recovery

% inhib.

Control

in vitro

% inhib.

times 90

% inhib.

51.8+0.5 51.5t2.8 _ 44:4+1.0 -

0 16

Slices were preincubated with the iodocompounds for 60 min and t.ransfered to fresh buffer for the indicated times(in min)for recovery.The T/M ratio was determined afterwards as described.Each value is the average of 4 slices -+SEM. In Vivo effects Treatment

Dose pg/day

Control

Table 4 on 1251 accumulation 1251 accumulation T/S rati. 254.0+ - 48.0 95.8: 15.0”

I-011-A

5

T3

5

46.5+

KI

1.25

185.4:

8.7* 6.0*

% Inhibit.

62 82 27

Rats were injected during 5 days as indicated.T/S ratio was measured 2 h after the administration of the label.Each value is the average of 4-5 animals+SEM.* : p< 0 .Ol when compared to the controls. DISCUSION The results herein presented confirm and extend our previous observations that iodinated derivatives of arachidonic acid exert a direct inhibitory effect on thyroid function(15-16).The present studies were performed with different purified compounds and allow us to draw conclusions concerning the relationship between structure and biological activity.The compound 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid(I-011-A) had the greatest inhibitory potency,when compared to its lactone or to the lactone bearing an iodine atom in the 6 position.It is interesting to mention that T3 failed to alter iodide uptake in vitro.Berkowitz et al(20) have shown that iodine inhibition of 1251 uptake is mediat-

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PROSTAGLANDINS

ed by an organic intermdiate.Our observation that T3 in vitro does , not mimic this effect of iodide would exclude this hormone as an intermediate,as suggested by Socolow et al(21) .Conversely,the iodinated derivatives that we had studied reproduce the effects of iodide on uptake,thus fulfilling one of tfre requirements to be consadered an intermediate in the autoregulatory mecfnanism.Berkowitz et al(20) Comparative

Exp.

effects

Treatment

A

Control

:,B

Control

Table 5 of the iodocomPounds % TCA precip. radioactiv.

I-al-A IL- w

on iodide

organification

P<

% Inhibition

41.8t1.2 8.3+1.1 -

0.001

80

67.3+4.5 25.6+1.9 _

0.01

62

0.05

37

AA

42.4+4.3 69.lt6.3 _

n.s.

0

1’3

66.5+5.1

n.s.

0

IL- $)

All compounds were tested slicesGEM. Effects

of

at 0.1 nN.Each value

Table 6 I-OH:A on basal and stimulated

Treatment

% TCA precip. radioactiv.

Control I-XXI-A 10-4M

56.7+2_2 16.350.3

IO-5M

p <

is the average

‘251 organification % Variation vs Control vs TSlI

0.01

- 72

34.3+1.5 52.921.5

0.01

- 40

n.s.

0

TSH 20 mu/ml

76.5i2.4 -

0.05

+ 35

TSH+I-Of+A IO-4M

20.4~2.4

0.01*

10_6M

Each value

of 4

- 74

10_5M 0.05* 62.Ot3.5 is the average of 4 slices+SEM.* -

vs TSfl alone.

- 19

have shown that iodide at 3 lo-‘M inhibited the T/M ratio by 21% after 30 min of preincubation,with a progressive inhibition of 37% at 75 min and 48% at 120 min,respectively.Our results demonstrate that preincubation with lo-5M I-OH-A causes an inhibition of the T/M ratio of 29% at 15 min and that longer times of preincubation lead to a Progressive decrease of this parameter(40% at 30 min and 54% at 60 min) .We may therefore conclude that the action of I-(X1-A is faster than that of iodide at similar concentrations .This action of iodide was reversed by tN1.

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In our studies,MMI did not alter the effect of I-Off-A, indicating that this compound exerts a direct action on the thyroid,and that the iodi de that would hypothetically arise from its dehalogenation is not responsible for the inhibition observed.It is also interesting to remark that the inhibition caused by I-Off-A is reversed after a recovery time of around 45 min.This iodocompound also inhibited iodide uptake in vivo,an effect attained with a dose of S ug/day.Since this iodocompound does not affect circulating TSff(22)we may again conclude that the action observed is due to the direct inhibition of the gland. When iodide organification was st.udied,again I-Ofl-A was the most potent inhibitior as compared to ILwand IL-$.Not only basal ,hut also TSH-stimulated 1251 organification was significantly inhibited by I-Off-A and this effect was dose-related.We have recently demonstrated that the inhibition of iodide organification caused by I-OH-A is due to a decreased ff202 availabi.lity ,without change in peroxi dase act ivi‘~(16). We may therefore conclude that different iodinated derivatives of arachidonic acid are inhibitors of iodide metabolism within the gland, and therefore mimic the action of iodide in thyroid autoregulation.

Nagataki S:Effect of excess quantities of iodide;in M.A.Greer E D.ft.Solomon(eds)Thyroid,flandbook of Physiology,sect.7,American Pfiysiological Society,Washington DC,329,1974 Greer M.A. 6 DeGroot L.J.:The effect of stable iodine on thyroid secretion in man.Metabolism 5 :682,1956 Pisarev M.A. ,DeGroot L.J. C, flati R. :KI and imidazole inhibition of TSff and cyclic AMP-induced thyroidal iodine secretion. Endocrinology 88:1217,1971 Pisarev M.A. 8, Aiello L.O.:Studies on the mechanism of action of KI on thyroid protein biosynthesis .Acta Endocrinol. (Kbh) 82 :298, 1976

Pisarev M.A.,Aiello L.O. 8 Kleiman de Pisarev D.L.:Action of KI, thyroxine and cyclic AMP on 3f1-uridine incorporation into thyroid RNA.Acta Endocrinol.(fCbh)83:313j1Q76 Van Sande J. & Dwnont J.E. :Effects of thyrotropin,prostaglandin Ef and iodide on cyclic 3 t 5’ AMP concentration in dog thyroid slices.Biochem.Biophys.Acta 313:320,1973 Rapoport B. ,West M. 8 Ingbar S.11. :On the mechanism of inhibition by iodine on the thyroid adentate cyclase response to thyrotropic hormone.Endocrinology 99:11,1976 Uchimura If. ,Cheng Chiu S. ,Kuzaya N. ,Ikeda II. ,Ito K. ,Nagataki S. : Effect of iodide enrichement in vitro on the adenylate cyclasecyclic 3’5’ adenosine monophosphate system in thyroid glands from normal subjects and patients with Graves’ disease.J.clin.Endocrinol. Metab. 50:1066,1980 Pisarev M.A. & Itoiz M.E.:Action of KI on stimulated thyroid protein biosynthesis.Endocrinology 90: 1409,1972

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10 Pisarev

M.A. :Thyroid

autoregulation.J.Endocrinol.Invest.8:475,

1985

M.A.,Juvenal G.J.,Kleiman de Pisarev D.L., 11 Chazenbalk G.D.,Pisarev Mercuri I-i. 4 DeTomas M. :Biosynthesis and regulation of iodolipids in calf thyroid.Acta Endocrinol. (Kbh) 108 :72,1985 12 Boeynaems J.M. 6 Hubbard W.C. :Transformation of arachidonic acid into iodolactone by the rat thyroid.J.biol.Chem. 255:9001,1980 13 Boeynames J.M. ,Reagan !.I. 6 IIubbard W.C. :Lactoperoxidase-catalyzed iodination of arachidonic acid:formation of macrolides. Lipids 16:246,1981 S.J. 6 IIubbard W.C. :Iodination 14 Turk J. ,fIenderson W.R. ,Klebanoff of arachidonic acid mediated eosinophil peroxidase ,myeloperoxidase and lactoperoxidase : identification and comparison of products. Biochim.Biophys.Acta. 751:189,1983 15 Chazenbalk G.D. ,Pisarev M.A. ,Krawiec L. ,Juvenal G.J. ,Burton G. i$ Valsecchi R.M. :In vitro inhibitory effects of an iodinated derivative of nrachidonic acid on calf thyroid.Acta Physiol .Pharmacol. Lat.amer. 34:367,1984 16 Krawiec L.,Chazenbalk G.D.,Puntarulo S.A.,Burton G. Boveris A., Valsecchi R.M. & Pisarev M.A. :The inhibition of PBl251 formation in calf thryoid caused by 14-iodo-15-hydroxy-eicosatrienoic acid is due to decreased II202 availability.Ilorm.Pletab.Res.20:86,19~8 17 Corda D. E Kohn L.D.:Phorbol myristate acetate inhibits.<-adrenergically but not thyrotropin-regulated functions in FRTL-5 rat thyroid cells.Endocrinology 120:1152,1987 18 Corey E.J. ,Niwa II. 6 Falk .J.R. :Selective epoxydation of eicosacis-5,8,11,14-tetranoic(arachidonic)acid and eicosa-cis-8,11,14trienoic acid.J.Amer.Chem.Soc. 101:586,1979 19 Bartlett P.A. 4 Myerson J.:Stereoselective epoxydation of acyclic olefinic carboxylic acid via iodolactonization.J.Amer.Chem.Soc. 100:3950,1978 20 Berkowitz M. ,Daughttridge D. 6 Sherwin J.R. :Autoregulation of the thyroid iodide transport:possible mediation by modifying sodium cotransport.Amer.J.Phvsiol. 240:E37.1981 21 Socolow-E. ,Dunlap D. ,Sobel R. E Ingbar S.fl. :A correlative study of the effect of iodide administration on thyroidal iodide and organic iodine content.Endocrinology 83:737,1968 22 Pisarev M.A. ,Burton G. ,Busse Grawitz P. ,Chazenbalk G.D. ,.Juvenal G.J. ,Kleiman de Pisarev D.L. ,Krawiec I,. 6 Valsecchi R.M. :Postreceptor events in growth control,in Frontiers in Thyroidology, G.Medeiros-Neto 6 E.Gaitan(eds) ,Plenum Medical Book Co ,New York, 1986,vol.l,p.125 Acknowledgements : To Miss Mariana B.Alegre and Mrs.Beatriz Alderete de Manto for expert technical assistance.This work was supported by grants from the Argerltine National Research Council(CONTCET). Reprints should be requested to:Dr.Mario Nuclear,CNEA,Av de1 Libertador 8250,1429

Editor:

172

H. Behrman

Received:

A.Pisarev,Division Rioquimica Buenos Aires,Argentina

6-24-87

AUGUST

Accepted:

6-28-88

1988 VOL. 36 NO. 2