Document MGRyOZgMe95Q0OqYGgOYLMJVV

'IZfitrogcnic Activity of DDT nalogs and Polychlorinated Bip** lyls Joel Bitman* and Helene C. Cecil Became of the geometric similarity of DDT to the synthetic estrogen, stilhctrol, DDT and 52 related compounds were tested in n sensitive estrogen assay in ruts. Estrogenic activity was evaluated using the tg.hr glycogen response of the immature rat uterus. Diphcnylmeihnne, diplv.-nylcthanc, and triphenylmethane compounds were active when a /> or p'-posi- lion was unoccupied or occupied by an hydroxy or methoxy group. Halide or alkyl groups in the p.p'- positions rendered the compounds inactive. Poly chlorinated biphenyls and polychlorinated tri phenyls, compounds which nrc environmental pol lutants of industrial origin. were estrogcnically active. Phcnolpluhalol and phcnolpfuhaletn. com pounds which are used as laxatives in drug prepara tions, were also cstrogcnically active since they con tain the appropriate p.p'-dihydroxy structures. Stereo models indicated that p.p'-dihydroxy com pounds of the active nuclei would have internudear distances of the hydroxyl groups which would ap proximate those of the natural steroidal estrogens and the synthetic stilbene estrogens. ' n 1945 Sofmssen published an excellent and comprehen sive 117-jnigc review of the synthetic estrogens and the - relation between their structure and activity. In the 25 years since Solmsscn's review, DDT, a chlorinated hydro carbon with a geometric similarity to the synthetic estrogens, lias b-.cn widely used throughout the world for pest control. The recent demonstration by Welch ft at. (1969) of the estro genic activity of o.p'-DDT and >ur own investigations (Bitman ri at., 1963) hnvc prompted us to investigate a series of DDT analogs, homologs, and structurally related compounds in nn attempt to determine relationships of structure to estro genic activity. METHODS We used the sensitive 18-hr glycogen response of the rat uterus ns n measure or estrogenic activity (Bitman et at., 1965). The potency of active compounds is reported in lerms of the minimal subcutaneous dose which will increase glycogen to a level significantly different from control. The 18-hr gjycogcrue response is illustrated in Figure 1, in which the dose-response curve for o,/'-DDT is represented. The steeper response line for glycogen, as compared to uterine weight, is readily apparent. * lest substances were dissolved in olive oil or an aqueous ethanol solution and injected subcutaneously at n screening dose rate of 8 mg per rat. Immature female Wistar rats (21-23 days old; 36- 48 g) were killed 18 hr after the injection; uteri wetc quickly excised, weighed, and analyzed for gtycogen by the unthrone procedure (Sciftcr cr at., 1950). Substances showing activity were tested further at dosage levels to 0.05 mg. Statistical comparisons were made using Student's t test with coircction for unequal group size. AlllkK MV(AT ION'S ' Table I: /,/>'-DDT, I.I,t-lHchloro-2,2-bis(/?-chlorophcnybcthanc; Tetrachloro-DOT, l,l,l,2-tetrachloro-2.2-bis(p-chloi ophenyDethone; /?./>' DFDT, I, I, l-U iehloro-2,2-bis(p>nuorophen>IX'thnnc; p.p'-Pcrthanc, U.I-tiichloio-2,2bi'fp-ethyIphcnylX'thanc; p.p'-Kelthanc, M.f-tricMoro-2,2bis(p-chlorophenyl)ethane; p.p'-DDTF, l,l,l-trifUtoro-2,2bis(/M:Moroplu*nyt)etHanc; p.p'-DDD, l,l-dichloro-2,2-bi$- (/> chlorojihcnyOcthanc; p.p'-DDfi, l,l-dichloro-2,2-bis- Anlmal Husbandry Research Division, Bcltsvillc, Md. 20705 To whom correspondence should hr addressed. f/y-chlorophcnyljethylcnc; p.^'-DDMU, J-chloro-2,2-bji- (p-chlorophcnyl)clhylcnc; and p,p'-DDA, 2,2-bis(/vchloro* phcnyljaccitc acid. Table II: o.p'-DDT, l,!,1-trichloro*2-(/-chlorophcnyl)-2- (o-chlorophcnyl)cthnne; u,p'-DDE, I,!-dichlofo-2-(/>-cltoro- phcnyl),2-(f-chlorophcnyl)ethylene; o.p'-DDMU, I-dloro- 2-(/>-chlorophcnyl), 2-(o*chlorophcnyl)cthylcnc; o,/>'-DDD, 1,1 -dichloro-2-(/7-chlorophvnyl),2-(o-chlorophcnyl)cthane; w.p'-DDD, 1,1 -dichloro-2-(p-chlorophcnyl),2-(/ii*chloro- phcnyl)clhnt.c and p,p'-Mcthoxychlor, l.l.J-trichloro-2,2-bs- (p-mcthoxyphenyl)ethanc. Table III: Compound 20. I,l-Diphenylmcthnne; 21, 1,1- DichIoro-l,I-diphenylmcthanc; 22, l,l-bis(/-broniophcnyl)- methane; 23, I -(phcnyl)-l -(p-hydroxyphenyl)niethane; 24, l,J-bis(/-hydroxyi>henyl)mcthane; 25, l-(/-hydroxyphcnyl)- l-(/>-methoxyphonyl)mcihanc; 26, l-(phcnyl)-Wp-rucihoxy- r-hydroxyphenyl)melhane; 27, l.I-bis(u-hydroxyphcnyl)- methane; 28, l,l-bis(0-hydroxy.i-chlorophcnyl)nicihnnc; 29, l-(phcnyl)-l-(p-chlorophenyl)*l-methanol: 30, l,I-bis(/> ehlorophenylM-melbanol,* i), 2,2 '-dihydroxybenzophenone; 32, 2,4-dihydroxybcnzophcnonc; 33. 4,4'-dihydroxybcnzo- phenonc; and 34, 2,2'-dhydroxy-4,4'-dimethoxybcnzophe- none. Sources of (he compounds used in this study were: Nos. 4, 5, 16----Rohm and Haas, Philadelphia; 9, 13--Dr. G. F. Fries, U.S. Dept. Apr., Bcltsvillc; 17--E. JC. Du Pont dc Nemours A Co., Inc., Wilmington; 18- Sigma Chemical Co., St. Louis; 24, 27, 35, 42--K Si K Laboratories, Inc., Plain- view, N.Y.; 38-- Eastman Kodak Co., Rochester; 43-53-- Monsanto Co., St. Louis. All other compounds were pur chased from the Aldrich Chemical Co., Inc., Milwaukee. Purity, as given by the manufacturers, was better than 99%. RESULTS AND DISCUSSION The natural estrogens are steroids which contain a phenolic ring A and an oxygen function at the Cn position, while the synthetic esuogens, which are stilbene derivatives, contain two phenolic rings (Figure 2), It is apparent that active cstrogcnicity is dependent upon the presence of nt least one phenolic hydroxy ring structure. In most estrogen tests these compounds are active in the micrograrn or submierogram range. , In contrast to this, the chlot mated hydrocarbon pesticides related to DDT arc only active as estrogens in milligram amounts, a 1000-fold difference. The DDT analogs nrc not phenolic, but they may give rise to aromatic phenolic sub stitution during metabolic conversions in the animal. The UC8 i. Aurt. roou cnr.M., vol. is. no. 6. 1970 MONS 082897 Figure 1. Dosc-rcsp'onsc relationship: uterine weight, (lycogcn, nnrf *m>'-DDT DDT analogs arc compounds of the diphcnylcthnnc type (Figure 2). Other analogs tested were compounds or the diplwnylmclhitne or triphenylmethane series. We have also examined polychlorinated biphenyls and polychlorinated (riphenyls, compounds which have become increasingly im plicated ns environmental pollutants of industrial origin. We have not included in this study any steroids, synthetic estrogens, or mili-cstrogcns of the stilbcnc structure, and have excluded almost all compounds of the coumarin, isoflavonc, anthracene, and phenanthrenc type. DINIKNYLEIHANE COMPOUNDS - /),/'*l*osl!lons Occupied by Halide or Alkyl. The com pounds evaluated in Table I me diphcnyicthanc derivatives in Table J. Diphcnylrfhitnc Compounds with p,p'-Positions Occupied by Halide or Alkyl Groups R x-c-<0>x R' Croups No. X K R' Name 1 Cl 2 Cl H -CCI, a/'*ddt Cl --ceil Tctrachloro-DDT 3 II -CCI, ap'-dfdt 4 CM.CH, H -ecu /j.p'-l'crlhane 5 Cl OH - CCI. p.p'-Kclthane 6 Cl H ~CF, A/i'-DDTr- 7 Cl H -- CMCI. p,p'-DDO 1 'Cl -CCI, p,p'`DDE - 9 Cl 10 a -CMC! a/`*odmu H* -COOH py-DOA M.E.D. minimum rfTeciivf doit. * t - in/irilvr. Activity M.E.D.* mg 4 4 l I* l 1` 1* I* 1 which the AP'-posilions arc occupied by halide or alkyl groups. Almost all were d.cvoid of estrogenic activity; />,/*' DDT (empd 1) and tctrnchloro-DDT (empd 2) exhibited a slight glycogenic response. It appears that halide or alkyl substitutions in the Ap'-positions were stable, and during me tabolism in the animal body, little if any p,p'-phcnolic hydroxy compounds are produced. p otp,p' Position Occupied by -- H or -- OCI!. When one of the para positions of the aromatic ring is substituted by a hy drogen or methoxy group, the compound exhibits estrogenic activity (Table II). Potency is of a low order of magnitude, being approximately 1000 times less active than compounds of the stilbcnc series, but is similar in potency to coumarin and isoflavonc estrogens (BickofT cl at., 1900). The minimum effective dose (MED) of dicthylstilbeslrol which elicited ft glycogen response was 0.1 pg, os compared to the most active compound of Table I), o,/>'-DDT, empd 11, whose MUD was 0.25 mgThe phenolic character of the natural and synthetic estro gens has demonstrated the dependency of cstrogcnicity upon the presence of a phenolic structure. The aromatic rings of the active compounds of Table II are open, i.e.t they have $ p~ or ^'-position occupied by ~M and may give rise to phenolic substitution during metabolism. There also appears to be a requirement for the ethane chain to be inert, i.e, either the tri* chloroL-ihanc (-CH-CClj) or the vinyl halide group (>CCClj) must also be present (cnipds 11, 12, 15). Thus, empds 14, 15, and 19, containing more reactive 2-carbon chain con figurations, arc inactive, even though one of the aromatic rings could be hydroxylated to the phenolic structure. We have concluded that rapid in viva metabolism of these compounds is tcsponsiblc for then lack of activity. In the stilbcstrol series (Solmsscn, 1945)and in (he coumarin series (Bickoff ci at., 1960) of estrogens, /,/'*dinicthoxy com pounds arc less active than comparable /,/>'*dihydroxy com pounds. In the. chloi inaird diphcnyicthanc series (Table II), the Ap'-melhoxy compound, mclhoxychloi (<mpd t'J) was S 6 8 ? 0 O SNOW Table 11. Diphcnylvi.ane Compounds with p- or /^Position Occupied oy --H or --OCHi Ro P^C-^P No. P || . H 12 II J) H 14 H 13 1! 16 H 17 OCHi 18 OCIfi 19 11 7 -~C1 a a a CI ii OCHi OCH, H > M.U.D. . minlmuni effective dose. 0 Cl Cl a Cl m*Cl H II H '-a *-Cl 111 m inactive. K H ... H H H H H H R' -CC1, --cell --CHCI --CHCI, -CHCI, --CCI, --CCI, --CCI, --CHO Name oy-DDT o.pr-DDE o.p'-DDMU e.p'-DDD m,p'-DDD f,l,t-Trichloro.?.2-bis(phcnyl)elhnc />,/' MeihoxycSlor Tech. Mcthoxychlor (p.p'- -4- o.p'-) 2,2-l)is(<7-chlorophenyl)acclaldchyde mg 0.23 4 8 1* P 1 4 1 P approximately as active as other compounds which might give rise to phenolic hydroxy substitution on metabolism. Tech* nic.il nuthoxychlor (empd 18), which may contain an <>,/>'* melhoxychtor, was four times more active than pure ptp'- mcthoxychlor. . A series of diphcnylmcthnnc compounds was examined to determine structural correlates of estrogenic activity (Table 111). Active compounds contained either one or two /vhy* droxy or p-methoxy groups (empds 23, 24, 23, 32, 33, 34). J. Table III. Dlphcnyhnclhanc Compounds; Denzophrnone Compounds p'0>c-p _ o i* P^C-^P No, P Croupi 7R Activity M.E.D.* R* mg Diphcnylmethane derivatives 20 H 21 H 22 Br 23 H 24 OH 23 OCH, 26 - OCH, 27* II 28' It 29 H 30 Cl 31 H H H Bf OH OH OH H H H CI Cl H HH Cl ct HH HH H\ H H 'H H 'H 11 H HH H OH H OH ... 1* P P 2 .. 1 4 P P P P I P Denzophenone derivatives 32 H 93 ' OH 34 OCH, OH OH OCH, 2 2 4 M.G.D. minimum effective doe. * I * inactive. *bii(^hy eroxyphenyl)methane, * bi*(-hydro*y, m-chtorophcnypnieihane. The most active compound was /7,/?'-dihydroxydiphinylmethane which elicited a glycogenic response at the I mg dose level. Solrnsscn (1943) reported activity for this cornpour d at the 100 mg level, but the differences in bioassay procedures could explain part of this difference in result. In the diphcnylcthanc series, compounds with a /7-hydrogen and a stable ethane chain u'erc metabolized to active estrogens, probably containing a /7-hydroxy structure, fn contrast, diphcnylmcthanc compounds with a /i-hydrogcn were not active, probably being metabolized rapidly at the methane linkage and excreted from the body. Denzophenone derivatives, which contain the more stable ketone structure at the methane carbon, were active if a p-hydroxy was present (Table III). DIPHENYL PROPANE COMPOUNDS; TRIPHENYL METHANE COMPOUNDS Two diphcnylpropanc compounds were active (empds 33 and 36), the /,/7'-hydroxy compound exhibiting much greater activity than a /s.p'-dimctho.xy compound. Dihydroxy di phenyl propane (empd 35) was as active as,/'-DDT. Solmssen (1943) found that this compound was active at a 100 mg dose level. Since the />,/7'-dihydroxy structure appeared to be the struc ture conferring activity, phcnolphthalol. n phenyl substituted diphenylmcthnnc compound containing />,/'-dihydroxy groups was tested. Phcnolphthalol was as potent as any compound of the types studied. Ring closure, as in phcnolphthalcin. resulted in a 20-fold loss in potency. These compounds are not known to have estrogenic activity and arc extensively used as laxatives in a number of drug preparations. In Table IV two miscellaneous derivatives which bear some relation to closed ring diphcnylmcthanc structur'd* are in cluded: fluoteuc and 9,10-dimethylnnthrnrenc. Doth of these compounds were inactive when tested at dose levels up to 8 mg per rat. BIPHENYL AND TRIPHF.NYL COMPOUNDS ^ Two hydroxy biphenyl compounds were active but only at the 4 and 8 mg dose levels (Table V). In a series of poly chlorinated biphenyls, (he compounds containing up to 48% chlorine were active. A:, judged from gle chromatograms, *110 J. AGR. POOD CHRM.. VOL. 18, NO. 6, 1970 MQNS 002899 .sx?6 rsiANf O-O: to.tX H-H: iu* @-i-c-@* JTI18INE u.tX u.sX c OICKtNtirtOVANI pIPHt NVl ITMANC O-O: H-H: t.zX io.?X `Q-c-P` DirHENVtMtlHANC '`''mX MX l.iPHEMrtMeTHANC *hO> *K2*0>* p wkcnyi TUtWIHYl B TMfHIMt 0-0: Aio 8 mX H-H: n*X AlOB loX is.oX A TO B 7.9 X t.lX 0-0: AtoC s.jX H-H: 7.0 X AtoC iojX lit X Figure 2. Structural formulae of estrogenic compounds these products sre crude mixtures containing a number of compounds. A polychlorinated triphenyl containing 42% chlorine was found to be more active* at a 1 mg dose level. COKkIJ.MIONS IlfiTWlIliN CHEMICAL STRUCTURE AND IliOLOGICAL ACTIVITY Sclmctcr (1946) and his coworkers (Fisher ct at., 19S2; Reading and Schucler, 1950) have theorized that a rather large, rigid, lipoid soluble molecular structure with two active hydrogen-bond forming groups located at an optimum dis tance of 14.5 A units ftom each other would be estrogenic. They further stated that potency is decreased as the distance between groups is decreased or increased. While DOT possesses o relatively large, rigid, lipoid soluble molecular constitution, it docs not present active hydrogen atoms at the hypothesized optimum distance of 14.5 A, how ever. The presence of the electronegative chlorine atoms in the /vp' oricnimions would prohibit the existence of active hydrogen. U these p,p' chlorine atoms were metabolized to groups possessing active hydrogen, the possibility of estrogen action would exist. The general lock of estrogenic activity of P,/'-l3DT analogs suggests that such metabolism docs not occur readily in the biologicRl situations studied thus far. Conversely, tint activity of o./r'-DDT raises interesting theoretical relationships between chemical constitution and estrogenic activity. The o,p' chlorine atoms are not at the hypothesized optimum distnnrc. The exact nature of the tlive estrogen structure arising from u.p'-DDT, if it is not n.p'-DlVI itself, might provide important information relating lo the spatial configuration of an active estrogen. Dividing Stercomodcls were constructed of many of the dive structures to determine whether consistent stereochemi cal factors were present. Jntcrmiclcar distances were mea sured and were given in Figure 2 for both O to O atoms of assumed dihydroxy compounds, and for the H to H distance of the hydroxyl groups. Interatomic distances for the Dreid- models of exit a nr and MiR.rnc writ- found lo be much No. 35 36 37 38 39 40 , Plphcnylpropnne "fld lrlplicnylniclsianc Compounds * Formula Name Activity M.U.D.* mg fH* MO@~i~>OH <*H, 2.2-Bis(p-hy* 0.23 droxyphen* y()propanc CMjCI ch,CH-ch, CM, CH,CI CX, 1.3-Dichloro2,2-bii(/7mclltoxy, m- methyl- phenyl)* propane 4 Phenol phthalol 0.2 Phenol- 4 phlhalcin Fluorene P 9,10-Dimcth ylanthra- CM, ccne minimum effective dose. 1 - inactive. t* 0 0 6 ? 9 0 SNOW - Tabic V. Diphenyl and Triphcnyl Compounds CK2> HM2> N. Name Activity M.E.D.* mg 41 I'O'.Diplienol 42 p.p'-ltiphcnol Polychlorinated 2,2'-Dihydroxydiphcnyl 4,4'-Dihydroxydiphcnyl 4 B Diphenyl (PCD) 4) PCII Aroclor 1221 21 % Chlorine 44 PCD Aroclor 1232 32% Cl 45 PCB Aroclor 1242 42% Cl 46 PCD Aroclor 1248 48% Cl 47 PCU Aroclor 1254 54% Cl 48 PCD Aroclor 1260' 60% Cl 49 PCD Aroclor 1262 62% Cl " 30 PCU Aroclor 1268 68% Cl 31 PCIt Aroclor 4465 60% PCII. 40% polychlori nated triphcnyl (PCT), B 8 B B 1* 1* I 1* 65% Cl 32 PCI Aidetor 544? 42% Cl $3 PCI Aroclor 5460 60% Cl 1 1 * M.H.D, - minimum cfTectivs dose. * I Inactive. inirtlf. f limn the J 4.5 A quoted by Keasling and Schuclcr (1950). The 10.9 A we found agree closely with the X-ray crystallographic data of Notion ct at. (1963, 1964), who found 10.95 A foi 17p*cstradiol. This discrepancy in interatomic dif ferences mny l>e related to the improved accuracy of the cur rent atomic models, when compared to those used in 1950. The diphcnylcthanc, diphcnylmcthanc, diphcnylpropanc, lriphen>lmcthnnc, biphenyl, and triphcnyl compounds all have interatomic distances of 9.4 to 10.3 A for the most likely O lo O substitutions. The H to H intcrnuclear distances of the hydroxyl groups ranee from 9.1 to 11.I A in/hese compounds. Doth (he 0--0 and H--H intcrnuclear distance?, therefore, are only slightly si,...ilcr Ilian corresponding bond distances in natural and synthetic estrogens. The structural observations regarding estrogenic activity in the compounds studied indicated (fiat activity is conferred when a p- orp'-position is unoccupied (--11), oris substituted by --OH or --OCH j. Halide, or alkyl groups, occupying^thc p,p'*posilions render the compounds estroceoKally inactive. A stable ethane chain was found to be necessary for activity, e.g., the trichloroclhanc or the inert vinyl halide group; if either C of the ethane chain bears an oxygen function (alco hol, aldehyde, or acid), the compound is metabolized ond no estrogenic activity is observed. Some polychlorinated bi phenyl and triphcnyl compounds exhibited estrogenic ictivity. Measurements of intcrnuclear distances of Dreidin} stcric models indicated that active sites would be 9-11 A apart, a range similar to those found in natural and synthetic estro gens, Quantitatively similar estrogenic activity was obtained with a series of diphcnylmcthanc or triphcnylmclhanc deriva tives which contained p-OH functions. Correlations of struc ture with tetivity suggest that the active estrogens derived from p^r'-annlogs of DDT arc p-phcnolic metabolites. LITERATURE CITED BickofT, E. M., Livingston. A. L.. Book, A. N.. Arch. tUoihcm. IS, 262(1950). Bitman,Cecil, )l. C., Harris, S. J., Fries, G. F., Science 162,371 (1968). Biiman, J.. Cecil, II. C., Mench, M. L., Wrenn, T. R., I httoerhf ohgy 76. 63 (1965). Fisher, A. L.. Kensling, It. H., Schuclcr, F. W., Proc. See. Kvp. Ola. Mat. 81,*439 (1952). Kcailing, ft. It. Schuclcr, F. IV.,/. Amer. Pharm. Ass. 39.87(1930). Norton, D. A., Knriha, G., Lu. C. T.. Acta Crytt. 16,89 (1963). Notion, I>. A., KoMha, G.. Lu. C T., Acta Cryst. 17, 77 (1964). Schuclcr, F. VV., Science 103, 221 (1946). Sciflcr, S., Dayton, S., Novic, D., Munlwyier, E., Arch. Dioehem, 25# 191 (1950). Soimssen, U. V.. Chem. Rev. 37, 481 (1943). Welch, R. M.. Levin, W., Conney, A. II.. Toxical. Appi. Pharmacol. 14, 338(1969). Receivedfar review June 25,1970. Accepter! August IJ, 1970. \ } MONS 0S^901 *112 i ac;it. rooD cirr.M.. vot. is. no, 6. 1070