[CANCER RESEARCH 44,184-189, January 1984]

Dependence on Exogenous Metabolic Activation for Induction of

Unscheduled DMA Synthesis in Syrian Hamster Embryo Cells by

Diethylstilbestrol and Related Compounds

Takeki Tsutsui, Gisela H. Degen, Dietmar Schiff mann, Annette Wong, Heiji Maizumi, John A. McLachlan, andJ. Carl Barrett1

National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [G. H. D., A. W., J. A. M., J. C. B.]; Nippon Dental University Tokyo, 1-9-10 Fujimi, Chiyoda-ku, Tokyo 102, Japan ¡T.T., H. M.]; and Institute for Pharmacology and Toxicology, University of Wuerzburg, Versbachier Strasse 9, D-8700Wuerzburg, Federal Republic of Germany [D. S.]

ABSTRACT

Diethylstilbestrol (DES) induces morphological and neoplastiatransformation of Syrian hamster embryo cells in vitro in theabsence of any measurable induction of gene mutations, whichis consistent with the lack of genotoxicity of DES in a number ofother assays. However, a few reports of a genotoxic activity ofDES in certain systems have been published. In order to understand these differences, we have investigated whether DESinduces unscheduled DNA synthesis (UDS) in Syrian hamsterembryo cells under the conditions which result in cell transformation and have examined the role of an exogenous metabolicactivation system on DES-induced UDS. DES, over a concentra

tion range of 1 to 10 /¿g/ml,failed to induce any detectable UDSin the cells, while other known transforming agents, including UVirradiation (6 to 24 J/sq m), benzo(a)pyrene (0.1 to 1.0 pg/ml),and aflatoxin B, (10 to 100 //g/ml), induced significant levels ofUDS. In contrast, UDS was induced in a dose-dependent mannerby DES (1 to 10 /¿g/ml)after addition of an Aroclor-induced rat

liver postmitochondrial supernatant fraction and other cofactorsfor exogenous metabolic activation. In order to probe the basisfor this alteration in UDS induction, the ability of structuralanalogues and metabolites of DES to induce UDS was examined.In the absence of exogenous activation, the only oxidative metabolite of DES detected in the presence of the cells was cis.cis-

dienestriol, which did not induce UDS by itself. In the presenceof exogenous activation, c/s,c/s-dienestrol and its trans.trans-

isomer induced UDS but not to a greater extent than DES. Withthe addition of the exogenous metabolizing system, increasedmetabolism of DES to c/s.c/s-dienestrol and additional polar

derivatives of DES or dienestrol, possibly hydroxylated derivatives, were observed. With exogenous metabolic activation, te-trafluoro-DES and hexestrol, which differ in their ability to be

peroxidatively metabolized to quinone and phenoxyradical intermediates, both induced UDS, although tetrafluorodiethylstilbes-

trol at 10 //g/ml stimulated a higher level of UDS. None of theDES-related compounds examined was active in the UDS assay

without exogenous metabolic activation, but all of the compounds can potentially form phenoxyradical intermediates by aperoxidase-mediated reaction. The compounds which can be

further oxidized to a quinone were most active in inducing UDS.These results are consistent with the hypothesis that this peroxidase-mediated pathway is important in the induction of UDS,

although secondary metabolites may also be involved. Furtherstudies on the nature of the reactive intermediates of DES which

' To whom requests for reprints should be addressed.

Received May 24,1983; accepted October 7,1983.

induce DNA damage are needed. However, this study demonstrates an important parameter in the genotoxicity of DES andpossibly provides as explanation for some of the conflictingresults regarding the DNA-damaging activity of DES. Further

studies are needed to determine the biological relevance of thegenotoxic activity of DES under these conditions.

INTRODUCTION

DES2 is known to cause cancer in humans (16) and experi

mental animals (18). Unlike many other known carcinogens, DESis inactive in several mutational assays with bacterial (14,23,34)and mammalian cells (3, 5, 13, 21). When induction of genemutation and neoplastia transformation were assayed concomi-tantly in the same cellular system, DES induced morphologicaland neoplastia transformation of Syrian hamster embryo cells inthe absence of any measurable gene mutations at 2 genetic loci(5, 29).

However, there are some reports which indicate genotoxiceffects of DES in certain test systems and seem to conflict withthe negative findings cited above. DES has been reported by 2groups to induce gene mutations in mouse lymphoma cells inthe presence of rat liver PMS (7, 10). Martin et al. (25) reportedthat DES induces UDS in HeLa cells treated in the presence ofrat liver PMS, while Althaus ef a/. (2) found no effect of DES onUDS in cultured rat hepatocytes. Rudiger et al. (34) reported thatDES and some of its metabolites induce sister chromatid exchanges in human fibroblasts in culture. Hill and Wolff (17)observed that DES induces sister chromatid exchanges in lymphocytes from pregnant and premenopausal women, but hadonly a small effect in lymphocytes from men and postmenopausalwomen. On the other hand, Abe and Sasaki (1) failed to observean effect of DES on sister chromatid exchanges in Chinesehamster cells. Conflicting results on the induction of chromosomeaberrations by DES also have been reported (1, 6, 8,9,19,20).

The Syrian hamster embryo cell transformation model represents an important system to determine the relevance of carcinogen-induced effects, because the endpoints of interest can be

correlated to the induction of neoplastia transformation in the

* The abbreviations used are: DES, diethylstilbestrol [primarily frans-isomer;

abbreviations for diethylstilbestrol and analogues follow the nomenclature of Metzler and McLachlan (32)]; PMS, postmitochondrial supernatant; UDS, unscheduledDIMA synthesis; Z.2-DIES, c/s.c/s-dienestrol (¿-dienestrol);E.E-DIES, trans.trans-dienestrol («-dienestrol); FBS. fetal bovine serum; PBS, phosphate-buffered saline[0.14 M NaCI, 0.9 rriM CaCI2, 0.5 mM MgCI2 6H;,O, 3 mu KCI, 8 FTIMNa2HP04, 1mm KH2PO, (pH 7.4)]; B(a)P, benzo(a)pyrene; HU. hydroxyurea; HEX. hexestrol;TF-DES, 3.5.3',5'-tetrafluorodiethylstilbestrol; E-OES, frans-diethylstilbestrol;

HPLC, high-pressure liquid chromatography; Z-DES, c/s-diethylstilbestrol; 1-OH-DES, 1-hydroxydiethylstilbestrol;3'-OH-DES, 3'-hydroxydiethylstilbestrol; HEPES,

4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid.

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DBS-induced DNA Damage

same cells (4). Since the induction of cell transformation by DEScan be measured in this stystem, we have examined some ofthe factors which might influence the genotoxic activity of DESin these cells. To this end, we have asked the following questions, (a) Can DNA damage be detected by the UDS assay incells treated with DES and other chemicals which induce celltransformation? (b) Is the induction of DNA damage modified byaddition of an exogenous metabolic activation system to thecells?

We observed that DES failed to induce UDS under the conditions that result in cell transformation, while other chemicalcarcinogens were active. However, UDS was induced by DESafter addition of exogenous metabolic activation, even thoughthese factors are not required for cell transformation. To furtherunderstand the basis for this alteration in activity, structuralanalogues of DES, which differ in their ability to be metabolizedby the major pathway of oxidative metabolism mediated by theSyrian hamster embryo cells (28), were examined for their abilityto induce UDS with and without exogenous metabolic activation.Also, the activities of Z.Z-DIES, the major metabolite of DES,and its isomer E,E,-DIES were determined. Finally, we have

compared the metabolism of DES by the cells alone to DESmetabolism by the exogenous metabolic activation system.

MATERIALS AND METHODS

Cells and Growth Medium. Cell cultures were established from 13-day-old embryos of Syrian hamsters, strain LVG:LAK (Lakeview Hamster

Colony, Chartes River Breeding Laboratories, Newfleld, N. J.). Primarycultures of pooled littermates were cryopreserved in liquid nitrogen,secondary cultures were initiated from frozen stocks, and all experimentswere performed with tertiary cultures in a humidified atmosphere with10%CO2inairat37°.

Culture medium used was IBR-modifled Dulbecco's Eagle's reinforced

medium (Grand Island Biological Co., Grand Island, N. Y.) supplementedwith sodium bicarbonate (3.7 g/liter), 10% (v/v) Hy-Clone FBS (Sterile

Systems Inc., Logan, Utah), penicillin (100 units/ml), and streptomycin(100 /

T. Tsutsuiet al.

humidified atmosphere with 10% CO2 in air. After 2 hr, tubes with theincubation mixture were cooled on ice and immediately extracted withdiethylether (3 times with a 2-fold volume). Extracts were combined,

evaporated under vacuum, and dissolved in 200 ^i methanol. Aliquots ofthese extracts were used for determining radioactivity and further analyzed by HPLC.

Methods for studying the metabolism of ["C]DES in Syrian hamster

embryo fibroblast cells have been described previously (12) and aresummarized briefly; tertiary cultures of nearly confluent Syrian hamsterembryo cells, grown on 10-cm culture dishes in complete medium, wereincubated with ["CJDES (5 ^g/ml) for 4 hr at 37° in a 10% CO2:air

atmosphere (12). The medium was removed from the dishes, and thecells were trypsinized, sonicated, and then pooled with the medium.Ethanol was added to a final concentration of 80% (v/v), precipitatedprotein was removed by filtering, and the soluble ethanolic fraction wasevaporated under vacuum. The extract was dissolved in methanol andfurther analyzed by HPLC.

HPLC analysis was performed using a liquid Chromatograph (WatersAssociates, Inc., Milford, Mass.) consisting of 2 pumps (M 6000 A), agradient programmer (M 660), an injector (WISP 710 A), an absorbancedetector (M440), a data module, a C,e reverse-phase columns eluted

with a linear methanohwater gradient. Compounds in the column eluatewere detected by absorbance at 254 nm, or by radioactivity detected infractions mixed with Biofluor scintillation cocktail (New England Nuclear,Boston, Mass.) in a Beckman LS 9000 scintillation counter (BeckmanInstruments, Norcross, Ga ). The vast majority of soluble radioactivitycompounds could be identified based on comparison of their retentiontimes with those of reference compounds (12).

RESULTS

Effects of DES on UDS in Syrian Hamster Embryo Cells inthe Absence and Presence of Exogenous Metabolic Activation System. When Syrian hamster embryo cells were treatedwith DES for 3 hr over the concentration range of 0.1 to 10 ngfml, induction of UDS was not detected (Chart 2A). Treatment ofthe cells with DES (0.1 to 10 M9/ml) for 24 hr also did not induceUDS. Higher doses of DES greatly reduced the cloning efficiencyof the cells (28) and, therefore, were not tested. In contrast,treatment of the cells with a physical carcinogen, UV (6 to 24 J/sq m), resulted in a dose-dependent increase in UDS (Chart 2B).

Furthermore, treatment of the cells with the chemical carcinogens B(a)P (0.1 to 1.0 /¿g/ml)for 3 hr, or aflatoxin B, (10 to 100ng/m\) for 1 hr, induced UDS (Chart 2, C and D).

In contrast to the negative findings without exogenous metabolism, incubation of Syrian hamster embryo cells with DES (10/tg/ml) for 3 hr in the presence of liver PMS from male ratspretreated with Aroclor resulted in induction of measurable UDS(Chart 3). Comparable results were obtained with PMS from ratlivers induced with phénobarbitaland benzoflavone. The extentof induction was dependent on the concentration of the PMSfraction added to the cells and the incubation time. MaximumUDS induction with Aroclor-induced rat livers was observed with5% PMS and a 2-hr incubation period (Chart 3). These conditions

were used in subsequent experiments with DES and its analogues.

Dose Dependence of UDS Induction in Syrian HamsterEmbryo Cells by DES and Related Compounds in the Presence of an Exogenous Metabolic Activation System. In thepresence of an exogenous metabolizing system, DES over theconcentration range of 1 to 10 ng/m\ induced a dose-dependent

increase in UDS in Syrian hamster embryo cells (Chart 4). Nosignificant difference was observed in the number of viable cells

9

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1 °

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1 3 10DES(yufl/ml)

40

30

20

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0.1 0.3 1.0 10 30 100Aflatoxin Bl(/ug/ml)

Chart 2. Induction of UDS by DES, UV, B(a)P. and aflatoxin B, in Syrian hamsterembryo cells without exogenous metabolic activation. The cells were treated withthe indicated dose of the chemical or UV irradiation, and the induction of UDS wasmeasured after treatment as described in "Materials and Methods." [3H)Thymidine(I'HJdThd) incorporation ±S.D. (oars) was plotted after subtraction of background

incorporation.

15

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8

02 5 10 20PMS(%)

0123Reaction timeChr)

Chart 3. Effect of exogenous metabolic activation system on the induction ofDOS by DES. Syrian hamster embryo cells were treated with DES (10 ¿ig/ml)withvarying amounts of PMS (A) for 3 hr or for varying reaction times, with 5% PMS(B), and UDS was measured as described in "Materials and Methods." Bars, S.D.

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Chart 4. Induction of UDS in Syrian hamster embryo cells by DES, relatedcompounds, and B(a)P in the presence of an exogenous metabolic activationsystem. The cells were treated with the indicated dose of compound, 5% PMS,and other cofactors for 2 hr, and UDS was measured as described in "Materialsand Methods." Bars, S.D.

in control or treated cultures. DES-related compounds were also

tested for the ability to induce UDS. In the absence of anexogenous metabolic activation system, none of the DES-related

compounds induced UDS, the same result observed with DES.In the presence of an exogenous metabolic activation system,TF-DES (1 to 10 Mg/ml) induced a dose-dependent increase in

UDS, which was similar to that observed with DES over the

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DES-induced DNA Damage

same concentration range, and HEX induced UDS but with apoor dose response (Chart 4).

Two other DES-related compounds were also tested. Z,Z-DIES and E.E-DIES both induced UDS. However, the effect ofZ.Z-DIES at the highest dose tested (10 ¿/g/ml)was greater thanthat of E.E-DIES at the same dose (Chart 4). Treatment withdoses of E.E-DIES greater than 3 ng/m\ failed to increase UDS.

B(a)P induced UDS to a greater level in the presence than inthe absence of an exogenous metabolic activation system, although poor dose response were observed with both conditions(Charts 2 and 4).

Metabolic Profile of DES in the Presence of Syrian HamsterEmbryo Cells Alone or with Rat Liver PMS. In this study, weconfirmed our earlier report (12) that Syrian hamster embryo cell

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Chart 5. HPLC profile of DES metabolites in Syrian hamster embryo fibroblastcell cultures. Cells were incubated with ["C]DES in complete medium and extractedwith ethanol as outlined in "Materials and Methods." The extracts were chromato-

graphed using an HPLC system. The separation was carried out on a Cia-Bondapakcolumn (250 x 0.4 mm; Waters Associates) with a 30-min linear gradient of methanolin water 38 to 82% at a flow rate of 1.5 ml/min. The effluent was fractionated, andradioactivity was determined in 0.45-ml fractions. Arrows, position of referencecompounds chromatographed under identical conditions: /, 4'-hydroxypropiophen-

one; //, DES-ketone; ///, E-DES; IV, Z.Z-DIES; and V, Z-DES.

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time (mm

Charte. HPLC profile of DES metabolites from an incubation with 5% PMSfraction. [UC]DES was incubated with reaction mixture as described in "Materialsand Methods." The extracted material was chromatographed on the HPLC unitdescribed in Chart 5. Separations were carried out on a ODS-Zorbax 5 column(DuPont, Wilmington, Del.) with a 30-min linear gradient of methanol in water 56 to88% at a flow rate of 1.0 ml/min. The effluent was collected in 0.3-min fractions,and radioactivity was determined. Arrows, position of reference compounds chromatographed under identical conditions. 1, 4'-hydroxypropiophenone; 2, 1-OH-DIES; 3, 1-OH-DES; 4, DES-ketone; 5, 3'-OH-DES; 6, Indenestrol A; 7, E-DES; 8,

Z,Z-DIES; and 9, Z-DES.

cultures oxidatively metabolized E-DES to Z,Z-DIES (Chart 5).After a 4-hr incubation, about 2% the DES was converted toZ.Z-DIES, although the majority of radioactive material coelutedwith the parent compound E-DES and its isomer Z-DES. Con

jugated glucuronides were also formed in the presence of thecells (Chart 5) which, after hydrolysis with 0-glucuronidase,showed the same composition of aglycones found in the non-congugated material (i.e., DES and very small amounts of Z,Z-

DIES).In the presence of rat liver PMS under the conditions used for

the UDS assay, DES was oxidatively metabolized to Z.Z-DIES.After a 2-hr incubation, about 40% of the DES was converted tothis metabolite. In addition to Z,Z-DIES, E-DES, and Z-DES, at

least 4 other minor peaks of radioactivity were observed in theHPLC chromatogram of the reaction mixture (Chart 6). Radioactivity was found to coelute with parent compound E-DES (Peakd) and its stereoisomer Z-DES (Peak a). The major metabolitefound in Peak c eluted with Z,Z-DIES. A small amount of radio

activity eluted in region e with the same retention time asIndenestrol A, a nonenzymatic cyclization product of Z.Z-DIES

(31). Minor radioactivity peaks f, g, and i eluted in positions ofreference compounds 3'-OH-DES, 1-OH-DES, and 1-hydroxy-

Z,Z-dienestrol, respectively. Peak b cochromatographed with

pseudodiethylstilbestrol; however, peak h could not be identifiedbased on comparison with reference compounds.

DISCUSSION

We have demonstrated previously that, in the absence of anexogenous metabolic activation system, DES can induce morphological and neoplastic transformation of Syrian hamster embryo cells (5, 29). Under the same conditions, no measurableinduction of gene mutation at 2 genetic loci in the same cellswas observed (5). The results presented in this communicationshow that no measurable induction of UDS occurs in the cells inthe absence of PMS. These results are consistent with our earlierconclusion that DES-induced cell transformation can occur in the

absence of detectable direct DNA damage by these assays.However, treatment of the cells with DES does result in inductionof numerical chromosome changes, which has the same cellcycle dependence and dose-response curve as the induction of

cell transformation (35).We have also demonstrated previously that Syrian hamster

embryo cells in culture oxidatively metabolize DES to Z,Z-DIES

(12). This metabolic conversion is believed to involve reactiveintermediates such as a phenoxyradical and quinone (31), andthe reaction is mediated by a variety of peroxidases, includingprostaglandin synthetase (11). The findings that indomethacininhibits the metabolism of DES in Syrian hamster embryo cellsand that arachadonic acid stimulates the oxidative metabolismof DES support the hypothesis that prostaglandin synthetasemediates the metabolism of DES in Syrian hamster embryo cells(11, 12). We have also reported that the ability of DES andrelated compounds to transform Syrian hamster embryo cells inculture correlates with the ability of these compounds to bemetabolized by a peroxidase-mediated oxidation (13, 29). These

results support a role for this metabolic pathway in the inductionof cell transformation by DES (29).

In contrast to the results discussed above, DES was found toinduce UDS in Syrian hamster embryo cells when added to thecells with a rat liver PMS activation system. Under optimal

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7. Tsutsui et al.

conditions, the extent of UDS induction in the presence of thisactivating system was similar to that observed with B(a)P andother chemical and physical carcinogens. Conflicting results (24,25, 33) on the ability of DES to induce UDS in other cell systemsin the presence of rat liver PMS may be due to the failure tooptimize the concentration of PMS or reaction time in the negative studies (24, 33).

In order to probe the basis for DES-induced UDS in hamster

cells in the presence of PMS, we have examined the ability ofstructural analogues and a metabolite (Z.Z-DIES) of DES to

induce UDS with and without exogenous metabolic activation. Inaddition, we have determined the metabolic profile of DES in thepresence of an exogenous metabolic activating system. FourDES-related compounds also induced UDS in the presence but

not the absence of an exogenous metabolic activation system(e.g., TF-DES, HEX, E.E-DIES, and Z.Z-DIES). All of the DES-

related compounds can potentially form a phenoxyradical intermediate via a peroxidase-mediated pathway, and this intermediate may bind to DMA and induce UDS. Only DES and TF-DES

can be further oxidized to the quinone (27), which also might bea reactive intermediate. The greater efficiency of DES and TF-

DES to induce UDS is consistent with the concept that thispathway is important in the induction of this effect.

The profile of DES metabolites was altered quantitatively and,possibly, qualitatively by the exogenous metabolizing system.Interestingly, the major product observed under these conditionswas Z.Z-DIES which is formed by a variety of peroxidativeenzymes. Cytochrome P-450 is known to possess peroxidativeactivity in addition to its well-known mixed-function oxidaseactivity (16). Significantly more Z.Z-DIES was formed in the

presence of rat liver PMS and cells than in Syrian hamster embryocells alone. In addition, at least 4 other metabolites were formedin the incubation mixture with rat liver PMS (Chart 6). It is notclear whether this is a qualitative difference in metabolism underthe different conditions or whether the ability to observe themetabolites is due to the greater extent of metabolism with anexogenous metabolizing system. We have not conclusively identified these new products; however, they appear to be morepolar derivatives of DES or Z.Z-DIES based on their elution time

in the Chromatographie system we have used. For example,Peak e comigrates with Indenestrol A, which is a cyclizationproduct of Z.Z-DIES (31). Peaks f and g elute in the position ofS'-OH-DES and 1-OH-DES, respectively. Peak i comigrates in

the same position as 1-hydroxy-c/s,c/s-dienestrol (ß-dienestrol).

These products can be formed through a further oxidation ofZ.Z-DIES by a mixed-function oxidase (31).

Metzler (31) has shown that 1-hydroxy-c/s,c/s-dienestrol has

alkylating potential. Thus, ft is possible that this metabolitecauses DNA damage which results in UDS induction. Furtherstudies are needed to elucidate the reactive intermediates whichinduce UDS and the metabolic pathway for their formation.Studies are currently in progress to definitively identify the metabolites of DES in the presence of PMS and to test their abilityto induce UDS. Since the mutagenicity of DES in yeast isenhanced by oxidizing agents (30), it will be interesting to compare the active metabolites of DES in this system to those formedin the presence of rat liver PMS to determine if common muta-genie derivatives of DES are found.

The finding that an exogenous metabolizing system is requiredfor the induction of UDS by DES demonstrates an importantvariable in the genotoxicity testing of DES. This finding may

provide an explanation for some of the seemingly contradictorystudies on the DNA-damaging effects of DES. In some studies

which have reported a positive effect of DES in an assay whichmeasures some form of DNA damage (for example, gene mutations, UDS, or sister chromatid exchange), PMS-mediated ex

ogenous metabolizing systems have been used (7, 10, 14).Studies in which DES has been reported as a DNA-damaging

agent in vivo (6, 20) or in lymphocytes in vitro of certain women(17) may be due to the metabolic activity of the cells at risk.

It is important to note that, while DES is activated by a ratliver microsomal preparation to a DNA-damaging intermediate(s),

this activity is not observed with intact liver cells. DES does notinduce UDS in primary hepatocytes in culture or gene mutationsin V-79 cells cocultivated with the hepatocytes (2, 14, 36). The

low peroxidative activity of intact liver compared to rat liver PMSmay be important in explaining this difference (28).

The activation of DES observed with microsome-mediated

metabolism may or may not be relevant to the in vivo activity ofDES. This is emphasized by the finding that DES can inducemorphological and neoplastic transformation in the absence ofany measurable induction of gene mutation (5) or UDS (thisreport). Further studies are in progress to determine if theincreased genotoxicity of DES in the presence of rat liver PMSis sufficient to cause DES-induced gene mutations and increase

cell transformation Syrian hamster embryo cells.In summary, our studies have demonstrated that DES can

induce neoplastic transformation of Syrian hamster cells withoutdirect DNA damage. DES will damage DNA if an exogenousmetabolic activation system is added to the cells. DES as wellas other carcinogens such as B(a)P are metabolized by Syrianhamster cells without exogenous metabolic activation; however,the metabolism of DES is altered qualitatively and possiblyquantitatively by a rat liver PMS activation system. Understanding the significance of these findings to DES-induced transfor

mation and carcinogenicity will require additional studies.

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6. Bishun, N., Forster, S., Vaierà , N., and Williams, D. C. The clastogenic effectsof diethylstilbestrol on ascitic tumor cells in vivo. Microbios. Lett., 73; 27-31,1980.

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14. Glatt, H. R., Metzler, M., and Oesch, F. Diethylstilbestrol and 11 derivatives amutagenicity study with Salmonella typhimurium. Mutât. Res., 67: 113-121,1979.

15. Herbst, A. L, Ufelder, H., and Poskanzer, D. Adenocarcinoma of the vagina:association of maternal stilbestrol therapy with tumor appearances in youngwomen. N. Engl. J. Med., 284: 878-881,1971.

16. Hildebraudt, A. G., and Roots, I. Reduced nicotinamide adenine dinucleotidephosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes. Arch.Biochem. Biophys., 171: 385-397, 1975.

17. Hill, A., and Wolff, S. Increased induction of sister chromatid exchange bydiethylstilbestrol in lymphocytes from pregnant and premenopausal women.Cancer Res., 42: 893-896,1982.

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19. Ishidate, M., Jr., and Odashima, S. Chromosome tests with 134 compoundson Chinese hamster cells in vitro—a screening for chemical carcinogens.Mutât.Res., 48: 337-354,1977.

20. Ivett, J. L., and Tice, R. R. Diethylstilbestrol-diphosphate induces chromosomalaberrations but not sister chromatid exchanges in murine bone marrow cellsin vivo. Environ. Mutagen., 3: 445-452, 1981.

21. «insella,A. R. Elimination of metabolic co-operation and the induction of sisterchromatid exchanges are not properties common to all promoting or co-carcinogenic agents. Carcinogenesis (Lond.), 3: 499-503,1982.

22. Kuroki, T., Malaveille, Drevon, C., Picolli, C., Macleod, M., and Selkirk, J. K.Critical importance of microsome concentration in mutagenesis assay with V-79 Chinese hamster cells. Mutât.Res., 63: 259-272, 1979.

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DES-induced DNA Damage

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JANUARY 1984 189

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1984;44:184-189. Cancer Res Takeki Tsutsui, Gisela H. Degen, Dietmar Schiffmann, et al. by Diethylstilbestrol and Related Compounds

Cellsof Unscheduled DNA Synthesis in Syrian Hamster Embryo Dependence on Exogenous Metabolic Activation for Induction

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