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Diethylstilbestrol: hormonal or chemical carcinogen?
TIPS-April 1982
174
cinogens. For chemical caminogens, eleo tmphilic teactivity of the carcinogenpef se or of a metabolite is considered a premqui site, aud tht carcinogenic pmceas ap)wamto be initiated by covalent bindiig of the electrophiieto criticalcellular macromolecules, presumablyDNA. Honnonal catcinogenr,
on the other hand, am assumed to lack chemical reactivity. and consequently. ate
Every one of us is u~voida~y exposed to certain phammcofogically active agents; estrogenic compounds belong to this ubiquitous group. Apan fmm their being endogenous to the female and male human organism, estrogens ate encountered exo~e~usiy in a variety of sottrees. e.g. in !oral contraceptives. in cosmetrcs, in residues tn meat. and in plants. many of which contain estrogenic compounds as natural constituents. In the past few years. however, concern has increased about the toxicity of estrogens, and in pamcular about their potential to cause cancer. One of the estrogenis compounds that contobuted cou&ierably IO that concern is diethyistilbestml (DES). HCstoryoCDEStlsr DES is a stilbene derivative (Fig. I) which was synthesized in 1938 in the laborarory 0” E. C. Dodds’. Asiue from being easily available and inexpensive. it was scan found to be an even mom Rowerful estrogen after oral ~minis~tion than the natural estrogen, esuadiol- l7/3. Its clinical application, commencing in 1938, was confined mainly to use as an estrogeri sub stitute and to treatment of prostatic cancer. Around 1948. it was reponed that DES might preveut miscarriage, and s&sequently DES was administered to an estimatedtotal of 2-4 million pregnant women with threatened abortion, mostly in the U.S.A. The nonmedical use of DES also started amuncl 1948. From that year on.
implants of DE.5 wete used to fatten chieken. in t957. it was reported that DES administered as an implant or with the diet ~agmwth~~~ingeff~t in beefcattle aad sheep. While, in the U.S.A., thr,use in poultry was already prohibited in l9S9 by the FDA, its use as a g~~hp~~t~t in steerswas continued until 1979, and it is estimatedthat BOW,of thecattle raisedin the U.S.A. has nxeived DES”.
Today, DES is almost abandoned and only legalized for a few specific medical ~~~~~ions, e.g. the treatment of prostatic :umors. One reason for its abandonment is the ovenvhelming evidence for its m~~~n~ity.
not able to bind covatentlyto DNA. The mob&e of DES itself is not chemically reactive. However, as is the case for m0st chemical carcinogens,reactivity may be acquired by metabolic alteration of the compound.
The first tepott on the caminogenicity of DES appeared in the literamre as early as 1938. Since that time, DES has been found carcinogenic in several animal species, affecting various organs (for reviews, see Refs. 4.5 and 6). The following are just a few examples of tumors observed after DES ~rn~is~ion: burns tumors in male and female C3H mice. testicular ttmrors in DAL& mice, mammary and pitui~ tumors in male and female AxC rats. renal tumors in intact and castrated mate and castrated female Syrian golden hamsters, ovarian tumots in dogs. and endometrialtumorsin squirt4 monkeys. Starting in 197I, reportsby Herbst and
his assoeiatess linked certain rn~i~nt tumors and nonmalignant abnormalities of y3
Studies of the oxidative metabolism of
DES in various animal species and in h~ans ffor review, see Ref. 7) have shown that virtually every pan of the DES molecule is affected by metabolic oaklation. The major pathways am depicted together with their putative metabolic intermediates in Fig. 2. They comprise aromaticand dicta hy~xy~t~. epoxidation of the olellnic double bond, and oxidation of the stiibenediil structure to Z,Zdienestmi. (For a complete metabolic scheme,see Ref. 7. ) Electrophilic reactivity can be anticipated for some of the spites and inteunediates.e.g. the atune oxide, alkene oxide, and the DES-@,<‘-semiquinooe and .quinone. In acco&mx with the formation of reactive metabolites am the numerous findings of radioactively labelled DES covalentlyboundto DNA andproteins in V&W. Similar binding has also been observed under various metabolic conditionsin vitro. e.g. with microsomesfiom different organs, and with peroxidasepreparations8.
+3-gjJOH I
2
tH3
Geuetk toxicity of DES Due to the formation of reactivemetabothe vagina and cervix observed in young lites and their ability to bind covalently to women to theirin utero exposure to DES. DNA, DES should be expected to give a The transplacental carciaogenicity and positive response in shotWenn tests ~mto~nicity of DES suggested by these designed to assay the genetic toxicity of findings for humans have subsequently chemical carcinogens. However, in the been observed in several mdent species. most popular bacterial system, which uses the reverse rn~ti~ of Sut~o~I~ff suchas mice, hamstersand rats. ryphimurium strains (Amestest). DES Hormonaf v. cl~mkal carc@gen could not be found mutagenic in studies One of the questions raised by the performed in several IaboMorics. even tumorigenic effects of DES concerns the under a variety of metabolic conditions*. mechanism of carcinogenicity. Is the car- On the other hand, positive findings have ci~nicity due to the ~ activity of been reportedfor DES in severalassaysys DES, or is DES acting as a chemical car- ternsusing mammalian celW. For exam. cinogen, or do both mechanisms act ple, DES induced unscheduled DNA together? The meehaniszz.s) by which synthesisin H&a cells, as well as mutahormones contribute to tumor formation is tions in a mouse lymphoma cell lii and not understood. but it is assumed that it dif- sister chromatid exchanges (SCE) in fers basically from that of chemical car- human fibroblasts. The potency of DES
to induce SCE exceeded that of benzo(a)pyrene. but was completely sup pressedby u-naphthoflavone. as inhibitor of drug metabolism, indicating that the genetic toxicity of DES is, in fact. due to metabolic activatiorP. DES has also been shown to cause morphol~gical and neoplastic transformation of Syrian hamster embryo cells at a frequency similar to that observed with benzo(a)pyrene. In contrast to benzo(a)pyrene. however, DES failed to induce somatic mutations at two genetis
loci (hypoxanthinsguaninc phosphw tibosyl uansferase awl sodium and potassiumdependent adenosine ttiphos phatase) under the conditionsof transformation’. One possibleexplanation is that DES-induced transformationresultsfrom a mutation at the chromosome level. The ability to causechromosomedamagessuch as aneuploidy. chromosome nondisjunction, and cholchicinelike effects has been reponed for DES.
the organ-directedtumorigenicityof DES. Several possible meclunisms ate conceivable: (a) DES might act as a tumorinitiating agent (chemical carcinogen) in many tissues.but tumorsmay only become manifest in estrogekresponsiveorgan3due to the promoting effect of DES (hormonal carcinogen);(b) alternativeI). DES ma> be qhzcitic preferentially activated b) enzymes in the target organ5 for the carcinogenic effect; of particular interest for this mechanism is the enzyme peroxida3e. which is found in high activities in all organsdependingon estrogensfor growth. and which can metabolize DES to DN.Abindingelectrophiles;(c) lastly. somereactive DES metabolites retain their ~uqea icity. as has been shown for example for DES-3,*oxide. and may thus accumulate in estrogen target organh. The aGlable data do not allow closer discrimination of these mechanisms, as high pzro~dau activity and estrogen receptorshare been found in all target organs of DES carcinogenicity thus far.
PossibkmechanismsofDES
caMtogenkity involving reactive nletabdites The evidence repDrIed thus f;v clearly indicates that DE!3 has, in addition to its estmgenic potential, the typical properties of a chemical carcinogen. The question persistsas to how the two chamcteristics. that of a hormone and that of a chemical carcinogen. function together lo result in
ImplicaUonsfor Ihe risk assessment of olhereslrugedcampomds
The genotoxicityofDES and it) nh111~to transform mammalian cells in rirro impI> that its carcinogenicily ik nut onl) 4 cow-
quence of its estrogcnicit~ but aIs0 of ilx metabolic activation. As chtrogens tar) widely in their chemicul s~uc~res. and because their structures determine their ~xt;isolic fate. different esropns must be
174
cinogens. For chemical caminogens, eleo tmphilic teactivity of the carcinogenpef se or of a metabolite is considered a premqui site, aud tht carcinogenic pmceas ap)wamto be initiated by covalent bindiig of the electrophiieto criticalcellular macromolecules, presumablyDNA. Honnonal catcinogenr,
on the other hand, am assumed to lack chemical reactivity. and consequently. ate
Every one of us is u~voida~y exposed to certain phammcofogically active agents; estrogenic compounds belong to this ubiquitous group. Apan fmm their being endogenous to the female and male human organism, estrogens ate encountered exo~e~usiy in a variety of sottrees. e.g. in !oral contraceptives. in cosmetrcs, in residues tn meat. and in plants. many of which contain estrogenic compounds as natural constituents. In the past few years. however, concern has increased about the toxicity of estrogens, and in pamcular about their potential to cause cancer. One of the estrogenis compounds that contobuted cou&ierably IO that concern is diethyistilbestml (DES). HCstoryoCDEStlsr DES is a stilbene derivative (Fig. I) which was synthesized in 1938 in the laborarory 0” E. C. Dodds’. Asiue from being easily available and inexpensive. it was scan found to be an even mom Rowerful estrogen after oral ~minis~tion than the natural estrogen, esuadiol- l7/3. Its clinical application, commencing in 1938, was confined mainly to use as an estrogeri sub stitute and to treatment of prostatic cancer. Around 1948. it was reponed that DES might preveut miscarriage, and s&sequently DES was administered to an estimatedtotal of 2-4 million pregnant women with threatened abortion, mostly in the U.S.A. The nonmedical use of DES also started amuncl 1948. From that year on.
implants of DE.5 wete used to fatten chieken. in t957. it was reported that DES administered as an implant or with the diet ~agmwth~~~ingeff~t in beefcattle aad sheep. While, in the U.S.A., thr,use in poultry was already prohibited in l9S9 by the FDA, its use as a g~~hp~~t~t in steerswas continued until 1979, and it is estimatedthat BOW,of thecattle raisedin the U.S.A. has nxeived DES”.
Today, DES is almost abandoned and only legalized for a few specific medical ~~~~~ions, e.g. the treatment of prostatic :umors. One reason for its abandonment is the ovenvhelming evidence for its m~~~n~ity.
not able to bind covatentlyto DNA. The mob&e of DES itself is not chemically reactive. However, as is the case for m0st chemical carcinogens,reactivity may be acquired by metabolic alteration of the compound.
The first tepott on the caminogenicity of DES appeared in the literamre as early as 1938. Since that time, DES has been found carcinogenic in several animal species, affecting various organs (for reviews, see Refs. 4.5 and 6). The following are just a few examples of tumors observed after DES ~rn~is~ion: burns tumors in male and female C3H mice. testicular ttmrors in DAL& mice, mammary and pitui~ tumors in male and female AxC rats. renal tumors in intact and castrated mate and castrated female Syrian golden hamsters, ovarian tumots in dogs. and endometrialtumorsin squirt4 monkeys. Starting in 197I, reportsby Herbst and
his assoeiatess linked certain rn~i~nt tumors and nonmalignant abnormalities of y3
Studies of the oxidative metabolism of
DES in various animal species and in h~ans ffor review, see Ref. 7) have shown that virtually every pan of the DES molecule is affected by metabolic oaklation. The major pathways am depicted together with their putative metabolic intermediates in Fig. 2. They comprise aromaticand dicta hy~xy~t~. epoxidation of the olellnic double bond, and oxidation of the stiibenediil structure to Z,Zdienestmi. (For a complete metabolic scheme,see Ref. 7. ) Electrophilic reactivity can be anticipated for some of the spites and inteunediates.e.g. the atune oxide, alkene oxide, and the DES-@,<‘-semiquinooe and .quinone. In acco&mx with the formation of reactive metabolites am the numerous findings of radioactively labelled DES covalentlyboundto DNA andproteins in V&W. Similar binding has also been observed under various metabolic conditionsin vitro. e.g. with microsomesfiom different organs, and with peroxidasepreparations8.
+3-gjJOH I
2
tH3
Geuetk toxicity of DES Due to the formation of reactivemetabothe vagina and cervix observed in young lites and their ability to bind covalently to women to theirin utero exposure to DES. DNA, DES should be expected to give a The transplacental carciaogenicity and positive response in shotWenn tests ~mto~nicity of DES suggested by these designed to assay the genetic toxicity of findings for humans have subsequently chemical carcinogens. However, in the been observed in several mdent species. most popular bacterial system, which uses the reverse rn~ti~ of Sut~o~I~ff suchas mice, hamstersand rats. ryphimurium strains (Amestest). DES Hormonaf v. cl~mkal carc@gen could not be found mutagenic in studies One of the questions raised by the performed in several IaboMorics. even tumorigenic effects of DES concerns the under a variety of metabolic conditions*. mechanism of carcinogenicity. Is the car- On the other hand, positive findings have ci~nicity due to the ~ activity of been reportedfor DES in severalassaysys DES, or is DES acting as a chemical car- ternsusing mammalian celW. For exam. cinogen, or do both mechanisms act ple, DES induced unscheduled DNA together? The meehaniszz.s) by which synthesisin H&a cells, as well as mutahormones contribute to tumor formation is tions in a mouse lymphoma cell lii and not understood. but it is assumed that it dif- sister chromatid exchanges (SCE) in fers basically from that of chemical car- human fibroblasts. The potency of DES
to induce SCE exceeded that of benzo(a)pyrene. but was completely sup pressedby u-naphthoflavone. as inhibitor of drug metabolism, indicating that the genetic toxicity of DES is, in fact. due to metabolic activatiorP. DES has also been shown to cause morphol~gical and neoplastic transformation of Syrian hamster embryo cells at a frequency similar to that observed with benzo(a)pyrene. In contrast to benzo(a)pyrene. however, DES failed to induce somatic mutations at two genetis
loci (hypoxanthinsguaninc phosphw tibosyl uansferase awl sodium and potassiumdependent adenosine ttiphos phatase) under the conditionsof transformation’. One possibleexplanation is that DES-induced transformationresultsfrom a mutation at the chromosome level. The ability to causechromosomedamagessuch as aneuploidy. chromosome nondisjunction, and cholchicinelike effects has been reponed for DES.
the organ-directedtumorigenicityof DES. Several possible meclunisms ate conceivable: (a) DES might act as a tumorinitiating agent (chemical carcinogen) in many tissues.but tumorsmay only become manifest in estrogekresponsiveorgan3due to the promoting effect of DES (hormonal carcinogen);(b) alternativeI). DES ma> be qhzcitic preferentially activated b) enzymes in the target organ5 for the carcinogenic effect; of particular interest for this mechanism is the enzyme peroxida3e. which is found in high activities in all organsdependingon estrogensfor growth. and which can metabolize DES to DN.Abindingelectrophiles;(c) lastly. somereactive DES metabolites retain their ~uqea icity. as has been shown for example for DES-3,*oxide. and may thus accumulate in estrogen target organh. The aGlable data do not allow closer discrimination of these mechanisms, as high pzro~dau activity and estrogen receptorshare been found in all target organs of DES carcinogenicity thus far.
PossibkmechanismsofDES
caMtogenkity involving reactive nletabdites The evidence repDrIed thus f;v clearly indicates that DE!3 has, in addition to its estmgenic potential, the typical properties of a chemical carcinogen. The question persistsas to how the two chamcteristics. that of a hormone and that of a chemical carcinogen. function together lo result in
ImplicaUonsfor Ihe risk assessment of olhereslrugedcampomds
The genotoxicityofDES and it) nh111~to transform mammalian cells in rirro impI> that its carcinogenicily ik nut onl) 4 cow-
quence of its estrogcnicit~ but aIs0 of ilx metabolic activation. As chtrogens tar) widely in their chemicul s~uc~res. and because their structures determine their ~xt;isolic fate. different esropns must be