Document 6RzrZKeE4kX2vVgaJLX1YLMYm

Fslrogcnic Activity of DDT Analogs and Polychlorinated Biphenyls i Joel Bit man* and Helene C. Cecil Because of the geometric similnrily of DDT to (he synthetic estrogen, slithestrol, DDT and 52 refuted 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. Diphenylmelhane, diphenylethanc, nnd triphenylmethane compounds w ere acme when a p- or /i'-position was unoccupied or occupied by an hydroxy or methoxy group. Halide or alky l groups in the /re positions rendered the compounds inactive. Poly chlorinated biphenyls and polychlorinated tri phenyls, compounds which are environmental pol lutants of industrial oiuiiii. were cstroeenicnlly active. Phcnolpluhalol anti phcnolphlhalem, com pounds which arc used as laxatives in drug prepara tions, were also eslrogenicallv 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 intcrnuclear distances of the hydroxy! groups which would ap proximate those of the natural steroidal estrogens and the synthetic stilbcnc estrogens. n 1945 Solmsscn published an excellent and comprehen (p-chloropbenyT)cthylcne; g.g'-DDMU, l-chloro-2,2-bis- I sive 117-page review of the sy nthetic estrogens and the relation between their structure and activity. In the (/>-chlorophcnyT)clhylcnc; and p,p'-DDA, 2,2 bis(/>-cHorophcnyl)accl.c acid. 25 years since Solmsscn's review, DDT, a chlorinated hydro Table II: e.g'-DDT, 1,1 ,l-trichloro-2-(/i-chlorophen , l)-2- carbon with n geometric similar ty to the synthetic estrogens, (o-chloropl cnyl)ethanc; o,p'-DDE, 1,1-dichloro-2-(/i-cnloro- hns been widely used throughoi t the world for pest control. phcnyl),2-(r'-chlorophcnyl)ctliylcric; o.p'-DOMU, 1 -c.doro- The recent demonstration by Welch cl a/. (1969) of the estro 2-(/i-cli!orophenyl), 2-(n-chlorophenyl)ethylenc; d./j'-DDD, genic activity of a.g'-DDT and our owm investigations (Uit- 1,1 - dichlor y - 2-{/i-chloro[ihenyT).2 -(r>- thlorophcnyllct tanc; man n nl.. I96S) have prompted us to investigate a series of iii,/j'-DDD. 1,1 -dichloro-2-(/i-chlorophcn> l),2-(m-d loro- DDT analogs, homologs, nnd structurally related compounds phcnyl)cthnnc and p./i'-Mcthoxychlor, 1,1,1 -trichloro-2, !-bis- in an attempt to determine relationships of structure to estro (/i-niethoxy phenyl )et hanc. genic activity. Table 111: Compound 20, 1,1 -Diphenylmelhane: 21. 1,1- METHODS Dichloro-I,!-diphenylmelhane ; 22, I,l-bis(/> bromophi ny 11methane; 23, l-(phenyT)-!-(p-hyclroxyphenyl)methane: 24, We used the sensitive 18-hr glycogen response of the rat 1,1 bis(/)-h>droxyphcnyi)mcthnnc; 25,1 -(/j-hydroxy plunvl)- uterus ns a measure of estrogenic activity (Hitman ei at., l-(/i-methoxy pheny lImcthanc; 26, 1 -(pliens l)-l (/wne tluixy - 1965). The potency of active compounds is reported in r?-liydroxyp!icnyT)mcthanc; 27, I ,l-bis(r-hydrox> pin in I), terms of the minimal subcutaneous dose which will increase methane; 28, 1 ,l-bis(rr-hydroxy-iM-chlorophcny Dmetli.ine ; glycogen to a level significantly different from' control. The 29, l-(phcnyl)-1-(/)-chlorophcny])-l-mcllmnol; 50, I ,l-l'o(/>- 18-hr glycogenic response is illustrated in Figure 1, in which chlorophcnyl)-] -methanol; 31, 2,2'-dihydroxy Ixuizoplienonc; the dose-icsponse curve for a.g'-DDT is represented. The 32, 2,4-dihy'droxybcnzophenone; 33, 4,4'-dihy droxy benro- sleeper response line for glycogen, as compared to uterine phenone; and 34, 2,2'-dihydroxy-4,4'-dirnethoxy Ix-nzophu- weight, is readily apparent. ' nonc. 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., Ileltsville; I7--C. K.. Du I'om de (21-25 days old; 56-48 p) were killed 18 hr after the injection; Nemours & Co., Inc., Wilmington; 18 --Sigma Chemical Co., uteri were quickly excised, weighed, nnd analyzed for glycogen St. Louis; 24, 27, 35, 42--K. <5: K Laboratories, Inc., IT,un- by the unthrone procedure (Seifter c; a!.. 1950). Substances view, N.Y.; 38--Eastman Kodak Co., Rochester; 43- 53 -- show ing activity were tested further at dosage levels to 0.05 Monsanto Co.. St. LotJis. 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 belter than 97%. AIIIIKI.STATIONS " Table I: />,/>' DDT, l.l,l-irich!oro-2,2-bis(p-ch!oro- pheny l)elhanc; T 'ctracli loro-DDT, 1 ,l,l,2-tclrachloro-2,2-bis- (/i chlorophenyTX'th.'ine; p./T-DFDT, l,I,l-trichloro-2,2-bis- (/>-fliiorophcnyT)clhnne; Per thane, 1,1,1 -trichloro-2,2- bis(/j-ethy IphcnylX'thanc; p.p'-Kell hanc, 1,1,1 -trichloro-2.2- bi'(/ichlorophen> Dethnne; p./j'-DDTF, 1,1,I-trifluoro-2,2- bis(p-t hlorophenylKthane; /i./i'-DDD, 1 ,l-dichloro-2,2-bis- (p-chlorophcnylXThanc; p.p'-DDE, 1,1 -dichloro-2,2-bis- Anininl Husbandry Research Division, Bcltsville, Md. 20705 * To whom correspondence should be addressed. RESULTS AND DISCUSSION The natural estrogens are steroids which com.on a phenolic ring A and an oxygen function at the Co position, while the synthetic estrogens, which are stilbcnc derivatives, loniain two phenolic rings (Eigurc 2). It is apparent tli.it anise cstrogcnicity is dependent upon the preseme of at liau one phenolic hydroxy ring structure. In most estrogen le a Uu c compounds arc active in the microgram or submii i o;< mi range. In contrast to this, the chlorinated hvdrocarhon pi -u ul. related to DDT arc only active as estrogens m ioiIo.i m nmounts, a 1000-fold difference. I he DDI an dogs ai, i.,.| phenolic, but they may give rive to aromatic phenol.e in stitution during metabolic convulsions in the animal. I in: HUH J. ACiR. rnoi) CttriM., VOL. is, NO. 6, 1970 DSW 027827 STLCOPCB4011789 0.1 0.2$ D.S 1 2 4 6 8 It) mg OP'DDT Figure 1. Dose-response relationship: uterine weight, glycogen, and o,/>'-DDT DDT analogs arc compounds or the diphcnylcthanc lype (Figure 2). Ollier analogs tested were compounds of the diphcnylmclhnne or Iriphcnylmelhane series. We have also examined polychlorinated biphenyls and polychlorinated tri phenyls, compounds which have become increasingly implicaled ns environmental pollutants of industrial origin. We have not included in ihis study any steroids, synthetic estrogens, or anti-estrogens of lhc stilbenc structure, and have excluded almost till compounds of the coumarin, isoflavonc, anthracene, and phcnanthrenc type. DIPIIENVLETHANE COMPOUNDS - p,p'-Positions Occupied.by Halide or Alkyl. The com pounds evaluated in Table l arc diphcnylcthanc derivatives in Table 1. Diphcnylcthanc Compounds with p,p'-Positions Occupied by Halide or Alkyl Croups R x-c^>x B' Groups No. X R R' Name 1 Cl H -CCI, /i.g'-DDT 2 Cl Cl -- CCI. Telrachloro-DDT 3F H --CCI, r,p' DFDT 4 CH.CH, H --CCI, /j./i'-l'crthane 5 Cl OH -CCI, /,/>'-Kclthane 6 Cl H -CF, />,/'-DDTF 7 Cl H --C1ICI, P./i'-DDD 8 'a --CCI, p.p'- DDF. 9 Cl = CHCI p.p'-DDMU 10 Cl H -- COOH />,//DDA * M.U.D. minimum effective dove. k I - inactive. Activity M.E.D.* mg 4 4 l I* 1* l* 1` I* I which the /'.//-positions arc occupied by halide or alkyl groups. Almost nil were devoid of estrogenic activity; p,p'DDT (empd I) and telrachloro-DDT (empd 2) exhibited a slight glycogenic response. It appears that halide or alkyl substitutions in the '-positions were stable, and during me tabolism in the animal body, little iT any /j,//-phenolic hydroxy compounds are produced. p- or p,p' Position Occupied by -- H or -- OCHj. 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 n low order of magnitude, being approximately 1000 times less active than compounds of the stilbenc series, but is similar in potency to coumarin and isoflavonc estrogens (BickofT rl a!., 1960). The minimum effective dose (MED) of dicthylstilbestrol which elicited a glycogen response was 0.1 gg, as compared to the most active compound of Table I!, o,//-DDT, empd 11, whose MED was 0.25 mg. The phenolic character of the natural and synthetic estro gens has demonstrated (he dependency of cstrogcnicity upon the presence of a phenolic structure. The aromatic rings of the active compounds of Table 11 arc open, i.r., they have a p- or //-position occupied by --H and may give rise to phenolic substitution during metabolism. There also appears to be a requirement for the ethane chain (o be inert, i.e, either (he Irichloroclhane (-CH-CCIj) or the vinyl halide group (>C = CCb) 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 hydroxylatcd to the phenolic structure. We have concluded that rapid in rirn metabolism of these compounds is responsible for their lack of activity. In the stilbcstrol series (Solmsscn, 1945) and in the coumarin series (Bickoff cl at., 1960) of estrogens, /i,/>'-dimclhoxy com pounds arc less active than comparable />,//-dihydroxy com pounds. In the chlorinated diphcnylcthanc series (Table II), (he /j.p'-mclhoxy compound, mcthoxychlor (empd 17) was J Ant? !rw\r\ftri* * *--. - DSW 0 2 7 8 2 8 STLCOPCB4011790 Table II. Dlphcnylctham: Compounds with p- or p'-I'ositlor Occupied by --H or --OCli, p,'>-c-'p R' No. P Groops p' 0 11 H 12 H --C1 a Cl Cl 13 H aa 14 It Cl Cl 15 H Cl m-Cl 16 H HH 17 OCH, OCH. H 18 OCH, OCH. H 19 H H o'-a O'Cl M.E.D. minimum effective dose l - inactive. R H H H H H H H R' --CCI, --CCI, =CHC1 --CHC1, --CHCI, --cct, --CCI. -CO, --CHO Name o.p'-DDT o,p'-DDE o.p'-DDMU o.p'-DDD m,p'-DDD I,l.l-Tricliloro-2,2-bisrphenyl)thane p,p'-Me(hoxychlor Tech. Mcthoxychlor tp.p'- -f o.p'-) 2,2-Bis(o-chlorophcnyl)acctaldehydc Activity mg 0.25 4 8 1` 1` 1 4 1 l* approximately as active as other c impounds which might give rise to phenolic hydroxy substitution on metabolism. Tech nical mcthoxychlor (empd 18), which may contain an o,p'mcthoxychtor, was four times more active than pure p,p'mclhoxychlor. DJPHENYLMETHANE COMPOUNDS; BENZOI'HENONE COMPOUNDS A series of diphcnylmcthanc compounds was examined to determine structural correlates of estrogenic activity (Table III). Active compounds contained either one or two p-hy droxy or /j-methoxy groups (empds 23, 24, 25, 32, 33, 34). _t Table III. Diphrnylmelhanc Compounds; Benzophenonc Compounds p'-c-^p 9 i- p'Q-c-Qp No. p Group* p' R ActMly M.E.D.* R' mg Diphenylmethane derivatives 20 H 21 H 22 Br 23 H 24 OH 25 OCH, 26 OCH, 27' H 28' H 29 H 30 a 31 H H H Br OH OH OH H H H a Cl H H Ii a Cl HH HH H\ H H 'H H 'H HH HH H OH H OH i i* i* 2 l 4 ! I* I1 1` I` 1` Beruophenone derivatives 32 H 33 ' OH 34 OCH, OH OH OCH, ... 2 2 4 1 M.E.D . -* minimum effective dose. 1 1 inactive. <biv(f>-hy* fxyphcnytjmctlianc. 4 bis to-hydroxy, m-riiloiophcnyl) methane. 10 J. agr. pood CD EM.. VOL. IS, NO 6, 1970 The most active compound was p.p'-dihydroxydiph. nyl- methanc which elicited a glycogenic response at the 1 mg dose level. Solmsscn (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 diphenylcthanc series, compounds with a p-bydrogen and a stable ethane chain were metabolized to active estrogens, probably containing a p-hydroxy structure. In contrast, di- phenylmcthane compounds with a p-hydrogen were not active, probably being metabolized rapidly at the methane linkage and excreted from the body. '" -' Benzophenonc derivatives, which contain the more stable ketone structure at the methane carbon, were active if a ^-hydroxy was present (Table HI). DIPHENYL PROPANE COMPOUNDS; TRIPHENYL METHANE COMPOUNDS Two diphcnylpropanc compounds were active (empds 35 and 36), the p,p'-hydroxy compound exhibiting much greater activity than a p.p'-dimethoxy compound. Dihydroxy di phenyl propane (empd 35) was as active ns n.p'-DDT. Solnissen (1945) found that this compound was active at a 100 mg dose level. Since the p,p'-dihydroxy structure appeared to be (he struc ture conferring activity, phenolphlhnlol, a phenyl substituted diphcnylmcthane compound containing p,p'-dihydroxy groups was tested. Phcnoiphthalol 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 diphcnylmelhnnc structures arc in cluded; fluorene and 9,10-dinicthylnnthraccne. Both of these compounds were inactive when tested at dose levels up to 8 mg per rat. DIPHENYL AND TRIPHENYL COMPOUNDS ' Two hydroxy biphenyl compounds were active hut only at the 4 and 8 mg dose levels (fable V). In a series of poly chlorinated biphenyls, the compounds containing up to 48% chlorine were active. As judged from glc chromatograms. OSW 027829 STLCOPCB4011791 tsrxANf it to.aX ; haX STIlltNE tz.tX imX -C-P- C diehenueoeane -% o DIEHCNYUTHANE DlfHENYlMETHANE WEHENYtMETHANE : mX o-te p-1}X \ : to.X mX AHp> 0 0>6A<0 a^h h P B iehehyi TAIEHENYl TtlfHENYt it Aro B .X : toaX A JOB u.zX ts.oX A TO B M X f.l X >: AtoC 4zX i: z.oX AtoC raaX tit X figure 2. Structural formulae of estrogenic compounds f these products arc crude mixtures containing a number of compounds. A polychlorinated diphenyl containing 42%-( chlorine was found to be more active, at a 1 mg dose level. 'v CORRELATIONS KLTWEEN CHEMICAL STRUCTURE AND BIOLOGICAL ACIIVITV Schuclcr (1946) and his coworkers (Fisher el at., 1952; Kcasling and Schuclcr, 1950) have theorized dial a rather large, rigid, lipoid soluble molecular structure with two active hydropen-bond forming groups located at an optimum dis tance of 14.5 K 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 rclnlivcly 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 (he /L/i'-oriciunlions 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'-DDF nnnlogs suggests that such metabolism docs not occur readily in the biological situations studied thus fac. Conversely, the activity of o.p'-DDT raises interesting theoretical relationships between chemical constitution and estrogenic activity. The v,p' chlorine atoms arc not at the hypothesized optimum distance. The exact nature of the active estrogen structure arising from n,p'-DDT, if it is not n.p'-DDT itself, might provide important information relating lo the spatial configuration of an active estrogen. Ilreiding Slcrcomodcls were constructed of many of the active structures to determine whether consistent stcrcochcmi<at Tudors were present. Intcrnuclcar 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 DreidIng models of cstranc and stilbene were found to be much L, * No. 35 36 Table IV. Diphcnylpropanc and Triphcnylmethane Compounds Formula Name Activity M.E.D.- mg hH-^0>oh CH, CHjCI CH, CHjCI CHJ 2,2-Bis(/vhy- 0.25 droxyphenyl)propanc 1,3-Dichloro2,2-bis(**xmethoxy, m-mclhylphcnylF propane 4 37 tCM-" Phenol- 0.2 phlhalol "L JT 38 . Phenol- 4 phthalein (grc= 39 Fluorene 1* 40 M.E.D. 9,10-Dimcthylanthraeene CHj minimum cOcciivc dose. 1 inactive. l* }. AGR, FOOD CHUM., VOL.. 18, NO. 1^70 UU Tabic V. Diphenyl an'! Triphcnyl Compounds M3> No. Name 41 o.o'-Biphcnol 2,2'-Dihydroxydiphenyl 42 /r,p'-Ujphcnol 4.4 '-Dihydroxydiphcnyl Polychlorinated Biphenyl (PCB) 41 pen Aroclor 1221 21 % Chlorine 44 PCB Aroclor 1232 32% Cl 45 PCB Aroclor 1242 42% (1 46 pen Aroclor 1248 48% ( 1 47 PCU Aroclor 1254 54% Cl 48 PCB Aroclor 1260 60% Cl 49 PCB Aroclor 1262 62% C! '50 PCI! Aroclor 1268 68 % Cl 51 1'CB Aroclor 4465 60% PCB. 40% polychlori nated triphcnyl (PCT). 65% Cl 52 PCT Aroclor 5442 42% C! 53 PCT Aroclor 5460 60% C! M.E.D. - minimum effective dose. `I inactive. Activity M.E.D.- mg 4 8 8 8 8 8 P P I* P P 1 I* smaller than the 14.5 A quoted by Kcasling and Schuclcr (1950). The 10.9 A we found agree closely with the X-ray crystallographic data oT Norton cr at. (1903, 1964). who found 10.95 A for 1 7f?-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 diphcnylcthanc, diphcnylmclhanc, diphcnylpropane, triphcnylmethanc, biphenyl, and iriphcnyl compounds all hRve 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 from9.1 to 11.1 A in Jhese compounds. Both the O--O and H--H internuclear distances, therefore, arc only slightly smaller than corresponding bond distances in natural and synthcticcstrogcns. The structural observations regarding estrogenic activity in the compounds studied indicated (hat activity is conferred when a p- or//-position is unoccupied (--H), or is substituted by --OH or --OCH,. Halide, or alkyl groups, occupying (he p,p'-positions render the compounds cstrogcnically inactive. "A stable ethane chain was found to be necessary for activity, e.g., the Irichlorocthanc 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 triphcnyl 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 scries of diphcnyimelhane or triphcnylmethanc 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 Bickoff. fc. M,, Livingston, A. L., Book, A. N,, Arch. Biochcm. 88, 262(1960). Bitman, }., Cecil, H. C., Harris, S. 3., Fries, G. F., Science 162, 371 (1968). Bitman, J., Cecil, H. C., Mench, M. L., Wrenn, T. R., I ndocrino/ogy 76. 6.1 (1965). Fisher, A. L.. Kcasling, H. H., Schuclcr, F. W., Pcoc. Soc. L.xp. Bio. Med. 81, 439 (1952). Kcasling. M. H.. Schuclcr. F. W., J. Amcr. Pharm. Ass. 39, 87 f 1950). Norton, D. A., Karlha. G., Lu. C. T., Acta Cryst. 16, 89 (1963) Norton, D. A.. Karlha, CL. Lu. C. T.. Acta Cryst. 17, 77 (1964). Schuclcr, F. W., Science 103, 221 (1946). Seiflcr, S., Dayton, S., Novic, B., Munlwylcr, E., Arch. Biochem. 25, 191 (1950). Solmsscn, U. V., Chem. Rec. 37, 481 (1945). Welch, R. M.. Levin, \V., Conney, A. H., Toxicol. Appl. Pharmocoi. 14, 358 (1969). Receivedfor review June 25, 1970. Accepted August 13, 1970. \ \\ \ \ /\ 11*2 J. Arm. POOD Cl I PM.. VOL. 18. NO. 0, 1970 ^ v OSW 0^7831 STLCOPCB4011793