Document ZwrJXNJ5MB3Zb5XOLm2yOQx0

Estrogenic Activity of DD^j\nalogs and Polychlorinated Bip^nyls .. Joel Bitman* and Helene C. Cecil Because of the geometric similarity of DDT to the synthetic estrogen, stilbcstrol, DDT and 52 related compounds were tested in a sensitive estrogen assay in rats. Estrogenic activity was evaluated using the 18-hr glycogen response of the immature rat uterus. Diphcnylmethanc, diphenylcthanc, and triphcnylmcthanc compounds were active when a p- or //-posi tion 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- phcnyls, compounds which arc environmental pol lutants of industrial origin, were cslrogcnically active. Phenolphthalol and phenolphthalein, com pounds which are used as laxatives in drug prepara tions, were also estrogenically active since they con tain the appropriate p./j'-dihydroxy structures. Stereo models indicated that p,p'-dihydroxy com pounds of the active nuclei would have internuclear distances of the hydroxyl groups which would ap proximate those of the natural steroidal estrogens and the synthetic stilbene estrogens. . n 1945 Solmssen published an excellent and comprehen (p-chlorophenyl)ethylene; p,p'-DDMU, l-chloro-2,2-bis- I sive 117-pagc review of the synthetic estrogens and the relation between their structure and activity. In the (p-chlorophcnyl)ethylene; and p,p'-DDA, 2,2-bis(/>-chloro- phenyl)acetic acid. . 25 years since Solmssen's review, DDT, a chlorinated hydro Table II: o,p'-DDT, l,l,l-trichloro-2-(p-chlorophenyl)-2- carbon with a geometric similarly to the synthetic estrogens, (o-chlorophenyl)ethane; o,p'-DDE, l,l-dichloro-2-(p-cMoro- has been widely used throughout the world for pest control. phenyl),2-(o-chlorophenyl)ethylene; o,p'-DDMU, 1-cl.loro- The recent demonstration by Welch et al. (1969) of the estro 2-(p-chlorophenyl), 2-(o-chlorophenyl)ethylcne; <?,p'-DDD, genic activity of o,p'-DDT and our own investigations (Bit- l, 1 -dichloro-2-(p-chlorophenyI), 2 - (o - chlorophcnyl)ethane; man et al., 1968) have prompted us to investigate a series of m, p'-DDD, 1 ,l-dichloro-2-(p-chlorophenyl),2-(i-chloro- DDT analogs, homologs, and structurally related compounds phenyl)eihar:e and p.p'-Mcthoxychlor, l,l,l-trichloro-2,2-bis- in an attempt to determine relationships of structure to estro (p-mcthoxyphenyl)cthanc. genic activity. Table III: Compound 20,1,1-Diphcnylmcthanc; 21,1,1- METHOD'S Dichloro-l,I-diphenyjmethane; 22, l,l-bis(p-bromophenyl)methane; 23, l-(phenyl)-l-(p-hydroxyphcnyI)methane; 24, We used the sensitive 18-hr glycogen response of the rat l,l-bis(p-hydroxyphenyl)methane; 25, 1 -(p-hydroxyphenyl)- uterus as a measure of estrogenic activity (Bitman et al., 1 -(p-methoxyphenyl)mcthane; 26, l-(phenyl)-l-(p-methoxy- 1965). The potency of active compounds is reported in o-hydroxyphenyl)methane; 27, l.l-bis(o-hydroxyphenyl)- terms of the minimal subcutaneous dose which will increase methane; 28, l,l-bis(<?-hydroxy-m-chIorophenyl)methane; glycogen to a level significantly different from control. The 29, l-(phenyI)-l-(p-chlorophenyl)-l-methanol; 30, I ,l-bis(p- 18-hr glycogenic response is illustrated in Figure 1, in which chlorophenyl)-l-methanol; 11, 2,2'-dihydroxybenzophenone; the dose-response curve for o,p'-DDT is represented. The 32, 2,4-dihydroxvbenzophenone; 33, 4,4'-dihydroxybenzo- steeper response line for glycogen, as compared to uterine phenone; and 34, 2,2'-dihydroxy-4,4'-dimethoxybenzophe- weight, is readily apparent. ' none. Test substances were dissolved in olive oil or an aqueous Sources of the compounds used in this study were: Nos. ethanol solution and injected subcutaneously at a screening 4, 5, 16--Rohm and Haas, Philadelphia; 9, 13--Dr. G. F. dose rate of 8 mg per rat. Immature female Wistar rats Fries, U.S. Dept. Agr., Beltsville; 17--E. K. Du Pont de (21-23 days old; 36-48 g) were killed 18 hr after the injection; Nemours & Co., Inc., Wilmington: 18--Sigma Chemical Co., uteri were quickly excised, weighed, and analyzed for glycogen St. Louis; 24, 27, 35, 42--K & K Laboratories, Inc., Plain- by the anthrone procedure (Seifter et al., 1950). Substances view, N.Y.; 38--Eastman Kodak Co., Rochester; 43-53-- showing activity were tested further at dosage levels to 0.05 Monsanto Co., St. Louis. All other compounds were pur mg. Statistical comparisons were made using Student's t test chased from the Aldrich Chemical Co., Inc., Milwaukee. with correction for unequal group size. Purity, as given by the manufacturers, was better than 99%. ABBREVIATIONS ,' Table 1: p,p'-DDT, l,l,l-trichlofo-2,2-bis(p-chlorophcnyl)e!hanc; Tetrachloro-DDT, l,l,l,2-tetrachloro-2,2-bis(p-chk>rophenyl)cthanc; p,//-DFDT, l,l,l-trichloro-2,2-bis(p-(luorophcnyl)cthane; p.p'-Perthane, l,l,l-trichloro-2,2bis(p-ethylphcnyl)cthane; p,p'-Kelthane, l,l,l-trichloro-2,2bis(p-chlorophenyl)ethane; p,p'-DDTF, l,l,l-trifluoro-2,2bis(p-chlorophenyl)ethane; p,p'-DDD, l,l-dichloro-2,2-bis(p-chlorophenyl)cthane; p.p'-DDE, l,l-dichloro-2,2-bis- Animal Husbandry Research Division, Beltsville, Md. 20705 To whom correspondence should be addressed. RESULTS AND DISCUSSION The natural estrogens are steroids which contain a phenolic ring A and an oxygen function at the C position, while the synthetic estrogens, which arc stilbene derivatives, contain two phenolic rings (Figure 2). It is apparent that active estrogenicily is dependent upon the presence of at least one phenolic hydroxy ring structure. In most estrogen tests these compounds are active in the microgram or submicrogram range. i In contrast to this, the chlorinated hydrocarbon pesticides related to DDT are only active as estrogens in milligram amounts, a 1000-fold difference. The DDT analogs are not phenolic, but they may give rise to aromatic phenolic sub stitution during metabolic conversions in the animal. The 1108 J. AGR. FOOD CHEM., VOL. 18, NO. 6, 1970 * DSW 373459 STLCOPCB4098947 I DDT analogs are compounds or the diphenylethane type (Figure 2). Other analogs tested were compounds of the di- phcnylmcthane or triphcnylmelhane series. We have also examined polychlorinated biphenyls and polychlorinated tri phenyls, compounds which have become increasingly im plicated as environmental pollutants of industrial origin. We have not included in this study any steroids, synthetic estrogens, or anti-estrogens of the stilbene structure, and have excluded almost all compounds of the coumarin, isoflavone, anthracene, and phenanthrene type. . DIPHENYLETHANE COMPOUNDS - p,p '-Positions Occupied by Halide or Alkyl. The com pounds evaluated in Table I are diphenylethane. derivatives in Table I. Diphenylethane Compounds with p,p '-Positions Occupied by Halide or Alkyl Groups R R' \ . "" - Groups No. X R R' \ Name 1 2 Cal H --CC1, p.p'-DDT Cl --CC1, 'Tetrachloro-DDT 3F H --CCI, p.p'-DFDT 4 CH.CH, H --CCI, p,p'-Perthane 5 Cl OH --CCI, p,p'-Kelthane 6 Cl 7a H --CF, p,p'-DDTF H --CHC1, p,p'-DDD 8 9 ''aCl . =CCI, p,p'-DDE , , , =CHC1 p,p'-DDMU . 10 Cl H --COOK p,p'-DDA . M.E.D. - minimum effective dose. 11 -- inactive. Activity M.E.D.* mg 4 4 1* l* 1* P I* I* I` 1* \t which the p,p '-positions are occupied by halide or alkyl groups. Almost all were devoid of estrogenic activity; p,p'DDT (empd 1) and tetrachloro-DDT (empd 2) exhibited a slight glycogenic response. It appears that halide or alkyl substitutions in the p,p'-positions were stable, and during me tabolism in the animal body, little if any p,p'-phenolic hydroxy compounds are produced. p- or p,p' Position Occupied by --H or --OCH,. 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 stilbene series, but is similar in potency to coumarin and isoflavone,estrogens (BickofT et a!., I960). The minimum effective dose (MED) of diethylslilbestrol which elicited a glycogen response was 0.1 pg, as compared to the most active .compound of Table II, o,p'-DUT, empd 11, whose MED was 0.25 mg. The phenolic character of the natural and synthetic estro gens has demonstrated the dependency of estrogenicity upon the presence of a phenolic structure. The aromatic rings of the active compounds of Table II are open, i.e., they have a p- or p'-position occupied by --H 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 trichloroethane (-CH-CClj) or the vinyl halide group (>C= CCh) must also be present (empds 11, 12, 13). Thus, empds 14, 15, and 19, containing more reactive 2-carbon chain con figurations, are inactive, even though one of the aromatic rings could be hydroxylated to the phenolic structure. We have concluded that rapid in vioo metabolism of these compounds is responsible for their lack of activity. In the stilbestrol series (Solmssen, 1945) and in the coumarin series (BickofT el al., 1960) of estrogens, p,p'-dimethoxy com pounds are less active than comparable p,p'-dihydroxy com pounds. In the chlorinated diphenylethane series (Table II), the p,p'-methoxy compound, methoxychlor (empd 17) was J. AGR, FOOD CHEM.. VOL. IS NO loin ItrtQ DSW 373460 STLCOPCB4098948 . Table II. Diphenyl ..Ine Compounds with p- or p '-Position Occupies J --H or --OCHi R p'<y \>)p *. . R' Groups ' No. P P' O 1112 H H 11115634 H '. H H H --aa aCal H aaaa m-Cl H 17 OCH, OCH, H 18 OCH, OCH, H 19 H H o'-Cl o-Cl M.E.D. - minimum effective dose. *1 -- inactive. R H H H H H H H R' --CC1, -=CCI, =CHC1 --CHd, --CHC1, --CCIi --CC1, --CC1, --CHO Name o,p'-DDT o,p'-DDE o,p'-DDMU ....... o,p'-DDD ,p'-DDD I,I,I-TrichIoro-2,2-bis(phenyl)ethane p,p'-Methoxychlor Tech. Methoxychlor (/,/'- + o,p'-) 2,2-Bis(o-chlorophenyl)acetaldehyde Activity M.E.D.* mg ` 40.2? 8 I 1 1* 4 1 I* approximately as active as other compounds which might give rise to phenolic hydroxy substitution on metabolism. Tech nical methoxychlor (cmpd 18), which may contain an o,p'methoxychlor, was four times more active than pure p,p'methoxychlor. DIPHENYLMETHANE COMPOUNDS; BENZOPHENONE COMPOUNDS A series of diphenylmethane compounds was examined to determine structural correlates of estrogenic activity (Table III). Active compounds contained either one or two p-hydroxy or p-methoxy groups (cmpds 23, 24, 25, 32, 33, 34). ---------------------------------------------------------------- 1 Table III. Diphenylmethane Compounds; Benzophenone Compounds R . P'<g>-C-<0>P O '* p*-4-p. No. P Groups p' R R' . Diphenylmethane derivatives 20 H 21 H 22 Br 23 H 24 OH 25 OCH, 26 - OCH, 27e_ H 28' H 29 30 Ha 31 H H H Br OH OH OH H H aH Cl H H Cl aH HH HH H H \H 'H H 'H HH HH H OH H OH .................... Activity M.E.D.* mg p p p 2-_ 1" 4 P P P I* P P Benzophenone derivatives 32 ; 33 ' 34 H OH OCH, OH OH OCH, aa aa. a .. 0 2 2 4 M.E.D. -- minimum effective dose. * I -- inactive. *bis(o-hydroxyphenyl)methane. * bis(o-hydroxy, m-chlorophenyl)methane. The most active compound was p,p'-dihydroxydi phenyl- methane which elicited a glycogenic response at the 1 mg dose level. Solmssen (1945) reported activity for this compound at the 100 mg level, but the differences in bioassay procedures could explain part of this difference in result. In the diphenylethane scries, compounds with a p-hydrogen and a stable ethane chain were metabolized to active estrogens, probably containing a p-hydroxy structure. In contrast, di phenylmethane compounds with a p-hydrogen were not active, probably being metabolized rapidly at the methane linkage and excreted from the body. ............. Benzophenone 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 diphenylpropane compounds were active (cmpds 3*5 and 36), the p,p'-hydroxy compound exhibiting much greater activity than a p,p'-dimethoxy compound. Dihydroxy di phenyl propane (cmpd 35) was as active as o,p'-DDT. Solms sen (1945) found that this compound was active at a 100 mg dose level. -' Since the p,p'-dihydroxy structure appeared to be the struc ture conferring activity, phcnolphthalol, a phenyl substituted diphenylmethane compound containing p,p'-dihydroxy groups was tested. Phenolphthalol was as potent as any compound of the types studied. Ring closure, as in phenolphthalein, resulted in a 20-fold loss in potency. These compounds are not known to have estrogenic activity and are extensively used as laxatives in a number of drug preparations. In Table IV two miscellaneous derivatives which bear some relation to closed ring diphenylmethane structures are in cluded: fluorene and 9,10-dimethylanthracene. Both of these compounds were inactive when tested at dose levels up to 8 mg per rat. BIPHENYL AND TRIPHENYL COMPOUNDS V 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, the compounds containing up to 48% chlorine were active. Az judged from glc chromatograms, 1110 J. AOR. FOOD CHBM., VOL. 18. NO. 6, 1970 DSW 373461 STLCOPCB4098949 , ' -f o , 0-0: H-H: kX iwX 9 ................. "....... ' ., ... Sill BENE imX hjX f C OIPHENYIEROPANE *-C-p- OIPHENYL ETHANE 0-0: .<* H--H: io.vX DIPHENYtMETHANE `* p`aaX mX |j) TBIPHENYLMETHANE A - . V* BIPHENYL 0-0: AtoB p.X H-H: km X TMPHENYl AtoB (4.2X is.oX TBIPHENYL AtoB pjX t.iX 0-0: AtoC 4.2X H-H: 7jo% AtoC kxsX ui* Figure 2. Structural formulae of estrogenic compounds these products are 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. CORRELATIONS BETWEEN CHEMICAL STRUCTURE AND BIOLOGICAL ACTIVITY ' Schueler (1946) and his coworkers (Fisher et at., 1952; Keasling and Schueler, 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 from each other would be estrogenic. They further stated that potency is decreased as the distance between groups is decreased or increased. While DDT possesses a relatively large, rigid, lipoid soluble molecular constitution, it does 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 /^.^'-orientations would prohibit the existence of active hydrogen. If these p,p' chlorine atoms were metabolized to groups possessing active hydrogen, the possibility of estrogen action would exist. The general lack of estrogenic activity of P,p'-DDT analogs suggests that such metabolism does not occur readily in the biological situations studied thus far. Conversely, the activity of o.p'-DDT raises interesting theoretical relationships between chemical constitution and estrogenic activity. The o,p' chlorine atoms are not at the hypothesized optimum distance. The exact nature of the active estrogen structure arising from o,p'-DDT, if it is not o,p'-DDT itself, might provide important information relating to the spatial configuration of an active estrogen. Drciding Stereomodels were constructed of many of the active structures to determine whether consistent stereochemi cal factors were present. Internuclear 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- ng models of estrane and stilbene were found to be much Table IV. Diphenylpropane and Triphenylmethane Compounds No. Formula Name Activity M.E.D.* mg 35 C", 2,2-Bis(p-hy- 0.25 HOH~>h droxyphen- yt)propanc 36 CHjCI .' CH, CHjCI CH3 1,3-Dichloro2,2-bis(/>- methoxy, m-methyl- phenyi)propane 4 37 .Phenol- . 0.2 phthalol 38 Phenol- 4 phthalein 39 Fluorene 1` 40 CH, CH, 9,10-Dimethylanthracene P * M.E.D. -- minimum effective dose. I -- inactive. DSW 373462 J. AGR. FOOD CHEM., VOL. 18, NO. 6. 1970 1111 STLCOPCB4098950  - - . V. J . Table V. Biphenyl and Triphenyl Compounds <OhO> ., No. Name 41 o.o'-Biphenol 2,2'-Dihydroxydiphenyl 42 p.p'-Biphenol 4,4'-Dihydroxydiphenyl Polychlorinated Biphenyl (PCB) 43 PCB Aroclor 1221 21% Chlorine - 44 PCB Aroclor 1232 32% Cl 43 PCB Aroclor 1242 42% Cl 46 PCB Aroclor 1248 48% Cl 47 PCB Aroclor 1254 54% Cl 48 PCB Aroclor 1260 60% Cl 49 PCB Aroclor 1262 62% Cl "30" PCB Aroclor 1268 68% Cl 51 PCB Aroclor 4465 60% PCB, 40%polychlori- nated triphenyl (PCT), 65% Cl 52 PCT Aroclor 5442 42% Cl .... 53 PCT Aroclor 5460 60% Cl * M.E.D. *= minimum effective dose. * I = inactive. Activity mg 4 8 8 8 8 8 P I` P P P 1 P smaller than the 14.5 A quoted by Keasling and Schueler (1950). The 10.9 A we found agree closely with the X-ray crystallographic data of Norton el al. (1963, 1964), who found 10.95 A for 170-estradiol. This discrepancy in interatomic dif ferences may be related to the improved accuracy of the cur rent atomic models, when compared to those used in 1950. The diphenylethane, diphenylmethane, diphenylpropane, triphenylmethane, biphenyl, and triphenyl compounds all have interatomic distances of 9.4 to 10.3 A for the most likely O to O substitutions. The H to H internuclear distances of the hydroxyl groups range from 9.1 to 11.1 A in Jhese compounds. Both the O--O and H--H internuclear distances', therefore, are only slightly i/^)er than corresponding bond distances in natural and synthetic estrogens. The structural observations regarding estrogenic activity in the compounds studied indicated that activity is conferred when a p- or p'-position is unoccupied (--H), or is substituted by --OH or --OCHj. Halide, or alkyl groups, occupying the p,p'-positions render the compounds estrogenically inactive. A stable ethane chain was found to be necessary for activity, e.g., the trichloroethane 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 and no estrogenic activity is observed. Some polychlorinated bi phenyl and triphenyl compounds exhibited estrogenic activity. Measurements of internuclear distances of Dreiding 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 diphenylmethane or triphenylmethane deriva tives which contained p-OH functions. Correlations of struc ture with activity suggest that the active estrogens derived from o,p'-analogs of DDT are p-phenolic metabolites. LITERATURE CITED . BickoflT, E. M., Livingston, A. L., Book, A. N., Arch. Biothem. 88, 262(1950). . Bitman, J., Cecil, H. C., Harris, S. J., Fries, G. F., Science 162, 371 (1968). Bitman, J., Cecil, H. C., Mench, M. L., Wrenn, T. R., E ndocrin ology 76, 63 (1965). Fisher, A. L., Keasling, H. H., Schueler, F. W., Proc. Soc. Exp. Bio. Med. 81, 439 (1952). Keasling, H. H,, Schueler, F. W.,/. Amer. Pharm. Ass. 39,87 (1950). Norton, D. A., Kartha, G., Lu, C. T., Ada Cryst, 16, 89 (1963). Norton, D. A., Kartha, G., Lu, C. T., Ada Cryst. 17, 77 (1964). Schueler, F. W,, Science 103, 221 (1946). Seifter, S., Dayton, S., Novic, B., Muntwyler, E., Arch. Biochem. 25, 191 (1950). Solmssen, U. V., Chem. Rev. 37, 481 (1945). Welch, R. M., Levin, W., Conney, A. H., Toxicol. Appl. Pharmacol. 14, 358 (1969). Receivedfor review June 25,1970. Accepted August 13, 1970. :\ ; -r V y V : \. \ \ % / \ /. \ / DSW 373463 1112 J. AGR. FOOD CHEM., VOL. 18, NO. 6, 1970 STLCOPCB4098951