Chem-BtoL lnteracturns, 76 (1990) 6 3 - 7 5 Elsewer Scmntlfic Pubhshers Ireland Ltd

63

INTERACTION OF CHLORINATED PHENOLS WITH THYROXINE BINDING SITES OF HUMAN TRANSTHYRETIN, ALBUMIN AND THYROID BINDING GLOBULIN

K J VAN DEN BERG

TNO Medzcal Bzologzcal Laboratory, Rz3sun3k fThe Netherlands] (Received February 12th, 1990) (Rewmon received May 27th, 1990) (Accepted May 29th, 1990)

SUMMARY

Previous results (Brouwer and van den Berg, Toxicol. Appl. Pharmacol., 85 {1986) 301) indicated preferential binding of a hydroxylated metaboIite of tetrachlorobiphenyl to transthyretin (TTR) a carrier of thyroxine (T4). In the present study it was investigated whether the T4 binding site of T T R could be occupied specifically by hydroxylated chlorinated aromatic compounds using chlorinated phenol congeners as model compounds in a competition assay with [12SI]T4.Chlorinated aromatics such as 2,3-dichlorobenzene and 3,4,3',4'-tetrachlorobiphenyl, and phenols such as 4-hydroxyblphenyl and phenol were inefficient competitors. All chlorinated phenols tested were competitors for the T4 binding site of TTR. The ranking in competition was pentachlorophenol (PCP) > trichlorophenols > dichlorophenols > monochlorophenols. Structures with chlorine m both ortho positions to the hydroxyl group were more efficient competitiors. The relative affinity of binding of pentachlorophenol (PCP) to T T R was about twice that of T4. Scatchard analysm showed that P C P mainly decreased the affinity constant K11 while the binding capacity R I was not altered, indicating a competitive type of inhibition. P C P was also able to compete with T4 sites on albumin with a relative affinity of 0.25. T4 binding to thyroid binding globulin (TBG) was much less affected by interference of P C P (relative affinity 0.001). The results indicate a specific interaction of chlorophenols with the T4 binding slte of TTR.

Key words. Chlorophenols -- P e n t a c h l o r o p h e n o l - T r a n s t h y r e t i n - Albumin - T h y r o i d binding globulin -- T h y r o x i n e binding site

Correspondence Dr K J van den Berg, Department of Occupational Tomcology, TNO Medical Biological Laboratory, P 0 Box 45, 2280 RIjswljk, The Netherlands 0009-2797/90/$03 50 © 1990 Elsevmr Scientific Publmhers Ireland Ltd Printed and Pubhshed m Ireland

64 INTRODUCTION It is well established that many environmental chemmals affect the morphology and hormonal status of the thyroid {reviewed in [1,2]} Particularly chlorinated aromatm hydrocarbons such as 2,3,7,8-tetrachloro-p-dloxm (TCDD), polychlorinated biphenyl (PCB), polybrommated blphenyl (PBB), hexachlorobenzene (HCB), and DDT have been found to disturb thyroid status In experimental ammals [3--10] Also in man the thyroid gland as well as the hormonal homeostasis may be altered as a consequence of exposure to chemicals such as DDD, HCB, PBB and organochlorme msectmldes [11--14]. The role of altered thyroid status in toxmlty by TCDD and related compounds is still under discussion [15] Recently, we have shown m marmoset monkeys that 3,4,3',4'-tetrachloroblphenyl (TCB) reduces histological and functional disturbances of the thyroid [16] m addition to a number of other toxic effects [17] Thyroid hypertrophy and hyperplasia was accompanied by reductions m plasma T4 and T3 levels and increased TSH levels [16] The cause of aberrations of the thyroid system by TCB and similar chemicals is not clear In the case of TCDD enhanced clearance of T4 may be revolved through strongly Increased hepatic mmrosomal metabohsm of thyroid hormones [3,18] The decrease in serum T3 and T4 by PCB or PBB has been attributed to both direct thyroldal and nonthyroidal damage rather than to enhanced hepatic or peripheral catabolism [19] It has been suggested [20,21] that the changes m plasma thyroid hormone levels by TCB may be caused by displacement of the physiological compounds from their binding sites on carriers. In the marmoset monkey [16] there was evidence for decreased T3 resin uptake indicating decreased availabhty of binding sites. Previously we have shown in rodents that a metabollte of TCB was assocmted specifically with transthyretin (TTR), a carrier of T4 [20]. McKinney and coworkers [22,23] have shown that a hydrophlhc derlvatlve of TCDD as well as several hydroxylated chlorinated PCBs and dlbenzofurans have affinity for the binding site of T4 on TTR and nuclear thyroid hormone receptors. These results suggest that hydroxylatmn of a halogenated aromatic compound may greatly facilitate interaction w~th TTR Early studies have indicated that both general binding as well as specific binding to the low affimty T4 site of albumin is higher for halogenated phenols as compared with phenols only [24,25]. Whether this property holds for the high affimty T4 rotes of TTR and thyroid binding globulin (TBG) is not clear In the present study the competitmn of various (chlorinated) phenol congeners with the T4 binding site of TTR was investigated In addition the relative and absolute affinities of PCP for the T4 binding site of the major human T4 transport carrmrs TTR, albumin and TBG [26] were determined

65 METHODS AND MATERIALS

Chemwals Chlorophenols and chlorobenzene were purchased from Aldmch (Brussels, Belgium), TCB was bought from Promochem (Germany). Stock solutmns of 0.023 M were made in ethanol except for TCB that was dissolved m ethanol/ DMSO (1:1). Dilutmns of the chemicals were made m ethanol. L-T4, human TTR and human serum albumin were obtained from Sigma (St. Lores, MO). Human TBG was from Calbiochem (Hoechst) (Germany). L-[3',5'-l~SI]T4 (spec. act. ~1500 ~Ci/~g, 55 MBq/~g) was obtained from Amersham (U.K.). All other chemicals used were of analytmal grade.

TI~ compet~twn assay The capacity of various compounds to compete with T4 binding sites was essentially as descmbed by Somack et al. [27] with modifmatlons. Bmefly, a 20-~1 ahquot of a solution of the compound m ethanol was added to 400 ~l phosphate buffered saline (PBS) consisting of 0 01 M phosphate (pH 7.3) and 0.154 M NaC1. Twenty microliters of radlolabelled T4 was added (about 0.2 ~C1/7.4 kBq) and subsequently 20 ~l TTR (1.1 pg/ml, final concentration). The reaction mixture was allowed to reach eqmhbrmm for 30 mm at room temperature. A 200-~1 ahquot of the reaction mixture was placed on a mml gelfiltration column kept at 0 °C The column was prepared m a 1 ml disposable syringe and consisted of a 1.00 _+ 0 05 ml bed volume of Sephadex G-25 m PBS The void volume of this column was 300 ~l TTR-bound radioactivity was eluted with 300 ~l me-cold PBS using slight overpressure to reduce transit time on the column (about 30 s) m order to minimize dlssocmtmn of the complex [27]. The column was subsequently eluted with 1 0 ml PBS to assess the amount of free iodide. Fresh batches of [125I]T4 contained about 10% free mdlde. Replicate 200 ~l samples were processed on separate columns. Radioactivity m eluate fractions was determined in a gamma counter. For determinatmn of maximum binding 20 ~l ethanol was added instead of the test compound Protein-bound radmactlvity amounted to 15% of the total amount of label m the reactmn m~xture. D~lutmns of albumin and TBG m the incubatmn mixture were chosen as to maintain this level of protein-bound radioact~vlty Non-specffm binding was also determined m each serms of expemments by addition of cold T4 to 10 ~M final concentratmn and was less than 10% for TTR and TBG and 15% for albumin. Percent competition was calculated for albumin carrmr after correctmn for non-speclfm binding

Scatchard analys~s The binding parameters were determined as descmbed above using cold T4 m concentrations ranging from 1 × 10 -s M to 4 × 10 -7 M [125I]T4 was added to maintain a constant specific activity of 3.5 × 10TM cpm/mol. In addltmn to protein-bound T4 and free red,de, non-protein bound that is retained

66

on the C-25 column, was determined by removing the gel from the mmlcolumn by slight overpressure and counting radioachwty In a g a m m a counter. Calculatlon of binding parameters was done wlth the L I G A N D - P C program [28] kindly prowded by Dr. P.J. Munson (NIH, Bethesda, MD). Significance of difference was tested using confidence ellipses [29]. RESULTS Previous results showed that in vlvo a hydroxylated metabolite of TCB (TCB-OH) was associated with TTR instead of the parent TCB [20,21]. This suggests that hydroxylatlon of chlorinated aromatic compounds may be important in the interaction with TTR. This assumption was tested wlth various chlorobenzenes and chlorophenols as model compounds. As analogues for TCB and TCB-OH were chosen in first instance 1,2-dlchlorobenzene and 2,3-dlchlorophenol (Fig. 1). Interaction with TTR and, more specifically, with the T4 binding site was determined with a rapid gel filtration method [27] The results (Table I) show that both TCB and 1,2-dlchlorobenzene at 100 ~M were inefficient compehtors for the T4 binding site of TTR. Neither phenol nor benzene at 100 ~M did appreciably interact with the T4 binding site. Also 4-hydroxyblphenyl at 100 ~M was a rather poor competitor. However, 2,3-dichlorophenol at 100/~M was a strong competitor for the T4 binding site. Higher chlorinated phenols such as 2,4,6-trlchlorophenol and PCP also appeared to be strong competitors. Insufficient amounts of TCB-OH were available for in vitro competition studies.

rl C I - ~

R2

R1 a1

a 2

TCB

H

3',4'-d~chlorophenyl

5-OH-TCB

OH

3',4'-d)chlorophenyl

1,2-Dichlorobenzene H

H

2,3-Dichlorophenol OH

H

Fig 1 General structure of model compounds

67 TABLE I COMPETITION OF SEVERAL AROMATIC COMPOUNDS FOR THE T4 BINDING SITE OF TTR Compounds were tested at 100 i~I m the standard competition assay as described m Materials and Methods Results are expressed as mean ± S D Compound

Competition (o~)

Benzene 1,2-Dlchlorobenzene 3,4,3',4'-TCB Phenol 4-Hydroxyblphenyl 2,3-Dlchlorophenyl 2,4,6-TCP PCP

0 0 11 10 14 87 91 96

± 4 ± 4 ± 3 ± 5 _+ 1 ± 2 ± 2 ± 1

In order to better differentiate between the interaction of chlorophenols congeners with TTR, competition curves were made over several decades of chlorophenol concentrations (Fig. 2). Chlorophenol congeners differed greatly in the abihty to interact with the T4 binding s,te of TTR Monochlorophenols having the weakest interaction and with increasing interaction followed by dichlorophenols and tnchlorophenols respectively. PCP showed the highest interaction with the T4 binding site. Hexachlorophene (2,2'-methylenbm(3,4,6-trichlorophenol)) was found to interact at a level in between PCP and the trichlorophenols. Within the dichlorophenols, 2,6-dlchlorophenol was a stronger competitor than 2,3-dichlorophenol. In the case of trlchlorophenols, 2,4,6-trichlorophenol dmplaced T4 more than did 2,4,5-trlchlorophenol. A maximum range in potency of interaction of chlorophenol congeners of about a factor 104 was observed. A compar,son in relative affinity of binding for the T4 binding site of TTR between the various chlorophenols and T4 was made on the basra of IC5o values. Table II shows that PCP had an almost 2-fold higher affinity for the T4 b,nding site than T4 itself. All other chlorophenols had lower binding afhmties than T4 with monochlorophenols having a relative affimty of only about 4 X I0-4. Another aim of thin study was to elucidate the mechamsm by which chlorinated phenols interfered wlth T4 binding to TTR e.g. by dlrect or redirect interference m the binding site. For thin purpose the binding characteristics of labelled T4 in the presence and absence of 50 nM PCP was studled according to Scatchard [30] using the "hot ligand" approach [28]. The graphic representation of the results (Fig. 3) shows that PCP merely affected the affinity of binding, leaving the number of binding sites largely unaltered. Quantitative analysis of the binding parameters showed a K1, of the control, e.g. 6 4 _+ 1.1 x 107 M -1, in good agreement with the value of 7.6 x 107 M-'

68 I

I

I

100

A ,u

-- BO cO

/

o

,,'"

60

,

/+

/./,:' I:

c]-

g

I

II /

×

;

u

I

/, albumin.

69 TABLE If AFFINITY OF C H L O R O P H E N O L S FOR T H E T4 BINDING SITE OF TTR ICe values determined from the competltloncurves shown in Fig 2 Competltlonexperlments wlth cold T4 were performed separatelyas describedm Materialsand Methods to demve the ICs0value for T4 Relatlveafflmtym expressedas: ICs L-thyroxine ICs testcompound Compound

ICs0(M)

Rel Affinity

PCP Hexachlorophene 2,4,6-TCP 2,4,5-TCP 2,6-D1chlorophenol 2,3-Dlchlorophenol 2-Chlorophenol

23 60 12 27 22 13 10

174 0 67 0 33 0 15 0 02 0 003 0 0004

× × x × × × ×

I0-8 I0-8 10-7 I0-7 10-6 10-5 10-~

3-Chlorophenol

1 4 × 10-'

0 0003

L-Thyroxine

4 0 × I0-8

10

DISCUSSION Previous results showed preferential association of a hydroxylated T C B over the parent T C B with T T R [20,21], a plasma carrmr for T4 and retinol binding protein. These results implied reductions in plasma levels of these physiological compounds to occur, but gave no direct evidence for e.g. occupation of the T 4 binding site. The present results demonstrate the capacity of relatively simple chlorophenols to interfere specifically with the T 4 binding site of T T R , albumin and T B G . Although a full account of quantitative structure activity (binding) relationships of chlorophenols will be presented elsewhere, the following qualitative properties were encountered. It appeared that a combination of a chlorinated aromatic ring and a hydroxyl group was a prerequisite for strong interaction with the T 4 binding site of T T R , either chemical property alone being less efficient. T h e degree of chlorination w a s also of importance since the higher chlorinated phenols showed a higher affinity for the T 4 binding site of T T R . P C P was found to be the strongest competitor for the T4 site of T T R , the relative affinity of binding being almost 2-fold higher than that of T4 itself. T h e position of the chlorine with respect to the hydroxyl group w a s also of importance. A structure with chlorine in oztho position to the hydroxyl group was a m o r e favourable configuration for interaction. These results taken together all are in close agreement wlth the general structural requirements of thyroxine and analogues for the binding s i t e of T T R [31].

70 0 50

I

\ 0

l

I

I

0

0 ~,0

rn

0 30

020 +PCP

0

~

010

0

z

I

1

2

i

Ao ~ . . ~

000 3 B (pmol T/.)

/+

Fig 3 Scatchard plot of competition of T4 binding to TTR by PCP Analysis of T4 binding to TTR m the absence and presence of PCP (50 nM) according to Scatchard [30] was performed as descr]bed m Methods and Materials

Scatchard analysis of the competition showed that the reduction of T4 binding to T T R by PCP mainly affected the association constant. This suggests that the mechamsm, by which displacement of T4 from TTR takes place, is probably through direct occupation of the T4 binding site by PCP In hne with this are the observations on the structural requirements. At this stage the binding of chlorophenol molecules to TTR, distal from the T4 binding site, cannot be excluded. In addition to the T4 binding sites of TTR, also simdar sites of human albumin and TBG can be occupied by PCP. The T4 binding sites of the latter are less well defined than the site of TTR that has been characterized extensively by X-ray diffraction [32]. PCP interacted appreciably with the T4 bradmg site of human albumin, in a gr eem ent with others [25]. The relative binding affinity of PCP (0 25) was found to be somewhat lower than report ed

71 1O0 90

oZ',,

80 70 c o Q_

E o (D

o~

60 50 40

30 20 10 0

..... i

........

i

........

J

........

i

........

i

........

i

........

i

t

.......

i

j

1 0 - ' 1 1 0 -1° 1 0 -9 1 0 -a 1 0 -7 1 0 -6 1 0 -~ 1 0 - "

(M) Fig 4 Competition of PCP with T4 binding site of albumin Ddferent concentrations of cold PCP (A--A) or T4 (O--O), as indicated on the x-axis, were incubated m the presence of [l~I]T4 and human serum albumin (174 ~g/ml) Competition was determined as described in Methods and Materials

[25]. The competition of PCP for the T4 binding site of TBG was, however, considerably less in comparison with TTR and albumin, having a relative affinity of only 0.001. This indicates that the T4 binding site of TBG is rather inaccessable to PCP. It also suggests that there may be significant differences in the configuration of the T4 binding sites between TTR and TBG. Quahtative and quantitative data on interaction of xenoblotics with TBG are notably scarce execpt for a few pharmaceuticals [26]. Marshall and Tompkins [11] have demonstrated binding of o,p'-DDD to TBG. Nuclear thyroid hormone receptor has been observed to bind halogenated xenoblotlcs. The interaction of monohydroxylated chlorinated aromatics with the nuclear thyroid hormone receptor occurred to a much lesser extent than with TTR [23]. The present paper describes for the hrst time the interaction of a single xenobiotic compound with the T4 site of the three major T4 plasma carriers m man. In relative affimties PCP has a selectivity for interaction with the T4 site of TTR. The selectivity of monohydroxylated TCB for general binding to TTR has been demonstrated by a quite different techmque [20,21].

72 100 o ~

90

/

80

/ /

70 C 0

Q_

60

E o L)

50

o~

40

/

/

/ /

z.,

30 20 10 0

°

..... J

10-"

....

/

~,,~

........

J ~

,

,,,.,,i

........

i

........

J

........

J

.......

a

,

1 0 -'0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 `4

(M) Fig 5 Competition of PCP vlth T4 binding site of TBG Different concentrations of cold PCP (A - A ) or T4 ( O - O ) , as mdmated on the x-axis, were Incubated in the presence of ['2SI]T4 and human TBG (130 ng/ml) Competition was determined as described in Methods and Materials

TABLE III AFFINITY OF PCP FOR T4 BINDING SITES OF TTR, ALBUMIN AND TBG ICs values for TTR as shown m Table II, values for albumin and TBG were determined from the data of F,gs 4 and 5 Carrmr

ICs0 (M}

Rel aff

Abs aff a (M-,)

T4

PCP

Albumin

4 0 X I0-s 4 0 x 10-8

2 3 x 10-s 1 0 x 10-7

17 0 25

12 X 108 1 7 x 105

TBG

15 x 10-9

1 5 x i0 -8

0001

i

TTR

x 107

•Affin,ty constants were calculated on basts of reported K values for T4 binding to TTR, Albumin and TBG 7 × 107 M -~, 7 x 105 M-' and 1 × 10 ~° M -j respectively [26]

73 From a toxicological point of vmw the present results point to the possible role of the hepatic cytochrome P-450 system in thyroid hormone effects of chlorinated aromatics. While the chlorinated aromatics themselves having comparatively little effect, conversmn by cytochrome P-450 into hydroxylated metabohtes might render these compounds more statable for interaction with T4 carriers. In vivo T4 plasma levels could thus be affected. There is now evidence that halogenated blphenyls that are known to alter thyroid hormone status such as PCBs and PBBs [4--7], might do so via the hydroxylated metabohtes [20,21,23]. Little information is available about direct effects of chlorinated benzene compounds concerning these msues. Hexachlorobenzene m known to alter thyroid morphology and to reduce plasma T4 levels both m man and m experimental animals [9,12]. PCP is one of the major metabohtes of HCB [33] and might be causing reduced thyroid levels through competition with T4 binding on carriers. Experiments are in progress specifically aimed at clarifying this further. Another toxicological implicatmn from the present results revolves the effects of human exposure with these compounds with respect to plasma thyroid status. Absolute affinities of PCP for the three plasma T4 carriers were estimated on the basra of the relative affmitms (Table III). The results suggest qualitatively that the effects on thyroid hormone status may differ in humans and m rodent expemmental models, because a TBG-hke carrier m lacking in the latter system Quantitatively, it may be possible to estimate the effect of PCP on human steady state plasma T4 levels with a computer model based on the law of mass actmn [34] and taking into account the absolute affimtms for the T4 carrmrs (m preparation). Finally, the present methodology offers a rapid manner of screening large numbers of chemmals for their potential effect on thyroid hormone status. At present, we have accumulated data of 74 chemicals, dmtributed over 13 chemical classes, for their interference m T4 binding to plasma carriers of which 58% were positive to varying degrees (m preparatmn). ACKNOWLEDGEMENTS

The author wishes to thank Prof. Dr. W.R.F. Notten, Drs. P.C. Bragt and G. de Mlk for comments and suggestions. REFERENCES 1 2 3 4

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23 24 25

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