The pesticide residues 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o1p'-DDT) and fi-hexachlorocyclohexane (@3-HCH) act asweak estrogens, producing uterotrophic responses in ovariectomized rodents and stimulating human breast cancer cells in culture. Such activitysuggests that these compounds may act as tumor promoters in estrogenresponsive tissues. Organochlorine compounds such as o@p'-DDTandfi-HCHareconcentratedin bodyfat.Thepresentreporttestswhethersufficient compound can be released from fat depots to produce estrogeniceffects in uteri of ovariectomized mice. Adult animals were “loaded―withtest compound by three daily injections of vehicle (DMSO), 17(J-estradiol(E2), @J-HCH,or o,p'-DDT.Uterotrophiceffectswereassessedat 24 hafter the last loading dose of test compound and at 2 weeks after theloading regimen, with or without a prior 2-day period of fasting. Theinitial 3-day treatment with either @3-HCH or o,p'-DDT doubled the
relative dry weight of the uterus: 102 ±8.6 mg/kg body weight (BW) and104 ±4.4 mg/kg BW for f@-HCHand oj?-DDT, respectively, compared to49 ±1.9 mg/kg BW for vehicle-treated animals. E2-treated animals haduterine dry weights of228 ±11 mglkg BW. After 2 weeks without furthertreatment, a 2-day fast produced a decrease in body mass of 4.1 g/animal(fasted, 25.9 ±1.89 g versus fed, 30.0 ±2.82 g). Animals that had beenloaded with @-HCHand fasted had uterine weights (88 ±12 mgfkg BW)significantly greater (P < 0.05) than those of vehicle-loaded, fasted ani
mals (51 ±2.9 mg/kg BW) or of @-HCH-loaded,fed animals (59 ±4.6mg/kg BW). The uterine weights of the fasted and fed o,p'-DDT-loaded orE2-loaded animals were not different from those of control weights. Thedifference between wet and dry weights showed that fasting of fi-HCHloaded animals also increased water imbibition in the uterus; there was noeffect from fasting in the other groups. Generally, epithelial cell heightreflected the same responses as uterine weight with the exception that cellheights of fJ-HCH-loaded, fed animals were slightly higher (P < 0.05) thancorresponding controls, indicating that there may have been some activecompound available to the tissues even without fasting. The effects offasting show that during periods of lipolysis @3-HCH can be released in
quantities sufficient to stimulate estrogen target tissues, suggesting a novelmechanism linking obesity and the progression of estrogen-responsivetumors. The lack of effect from fasting in o@p'-DDT-loadedanimals mdicates that these compounds are differentially mobilized from fat depots.
INTRODUCTION
Estrogens have been implicated in the etiology of benign andneoplastic tumors of the uterus and breast (1—3).Increased risk forthese hormonally sensitive cancers is associated with obesity, anovulatory infertility, late menopause, polycystic ovary syndrome, andsteroid-secreting ovarian tumors; increased risk of endometrial carcinoma is also associated with the use of estrogen as postmenopausalhormonal replacement therapy (1, 2). The basis of each of these riskfactors is believed to be an increase in circulating estrogen levels. Inthe case of obesity, the higher levels of estrogen that contribute to
Received 8/20/96; accepted 1/4/97.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.
t To whom requests for reprints should be addressed. at Obstetrics/Gynecology Re
search Laboratories, 1001 Walnut Street (MF 102), Indiana University School of Mcdicine, Indianapolis, IN 46202-5 196.
increased risk are believed to derive from conversion of adrenalandrogens to estrogens in fat cells (4, 5).
Epidemiological data suggest that there was a climbing incidence ofuterine cancer from the mid-l940s to the l980s (6); incidence ofbreast cancer has also continued to increase during the past severaldecades (7, 8). Although the acceleration in incidence of uterinecancers between 1968 and 1980 has been attributed to the increaseduse of estrogen replacement therapy in postmenopause (6), this cannotexplain the progressive increase in incidence seen during the earlieryears. [The widespread use of estrogens and progestins confoundsepidemiological data of recent years, particularly since progestinsprotect against endometrial cancer (I , 9).] Increased total lifetimeexposure to endogenous estrogens due to early menarche, age at firstfull-term pregnancy, age at menopause, and postmenopausal obesityhas been proposed as the basis for the increased incidence of breastcancer over the past 50 years (6). However, international differencesin incidence patterns cannot be fully accounted for by such a hypothesis, suggesting that established risk factors do not explain the increase in breast cancer incidence (7).
It has been suggested that increased incidence of endometrial andbreast cancers are linked to environmental factors (10—13).Man-madeestrogenic compounds are likely contributors to such an environmental effect. Several organochlorine compounds, including pesticideresidues and hydroxylated PCBs,2 have been characterized as estrogenic (14, 15). These xenoestrogens persist in the environment, haveentered the food chain (14, 16—18),and have produced dramaticendocrine-disruptive effects in wildlife (14). Furthermore, most organochlorine pesticides are fat soluble, facilitating their tendency topersist in the environment, bioaccumulate in the food chain, andconcentrate in human tissues, although their use has been banned indeveloped countries for more than two decades (1 1, 17—22).
The present report tests the hypothesis that xenoestrogens stored inbody fat will be released during periods of fasting and that thisreleased material will induce estrogenic effects in the uterus. Ovariectomized mice had their fat loaded with the prototypical xenoestrogen o,p'-DDT) (23) or with @-HCH,a less well-studied compoundwith weak estrogenic activity in the mouse uterus (24). It was foundthat uteri were stimulated following fast-induced hipolysis in !3-HCHloaded animals but not in o,p'-DDT-loaded animals. Such a findingsuggests a novel mechanism linking obesity with cancers of hormonally sensitive tissues.
MATERIALS AND METHODS
All procedures involving animals were approved by the Institutional Animal
Use and Care Committee. Adult ICR mice (25—30 g BW) were ovariectomized
under general anesthesia (ketamine). Groups of animals were left untreated for3 weeks and then given three daily injections of 5 nglg BW E2, 100 @.tg/gBWo,p'-DDT, or 100 @.Lg/gBW f3-HCH.Compounds were dissolved in DMSO anddelivered i.p. in 100 p1. Control animals received an injection of DMSO.
Groups of animals were sacrificed by cervical dislocation on the day following
2 The abbreviations used are: PCB, polychlorinated biphenyls; BW, body weight:
Xenobiotics Released from Fat during Fasting Produce Estrogenic Effects in
Ovariectomized Mice
Robert M. Bigsby,' Andrea Caperell-Grant, and Burra V. MadhukarDepartments of Obstetrics & Gynecology & Physiology & Biophysics, indiana University School of Medicine, Indianapolis, Indiana 46202-5196 fR. M. B., A. C.G.J, and
Department of Pediatrics and Human Development. Michigan State University, East Lansing, Michigan 48824 [B. V. M.J
the last injection of test compound. Other animals were ovariectomized, treatedwith compound, and then left untreated for 2 weeks. These animals were then
split into groups which were either fasted for 48 h or fed ad libitum as usual,
and then they were sacrificed as described above. All animals were weighed atthe time of sacrifice.
One uterine horn from each animal was processed for histological examination. It was fixed in buffered formalin, embedded in paraffin, and cut into6-p@msections. The sections were stained with H&E, and the height of theluminal epithelium was determined with the aid of an image analysis system
(IPLab Spectrum; Signal Analytics Corp., Vienna, VA); the height of 75—100cells was assessed for each specimen. The other horn was used to determine
wet and dry weights. This horn was dissected out of each animal, trimmed ofmesentery, slit longitudinally, blotted to remove luminal fluid, and thenweighed. After weighing, the tissues were desiccated in a 65°Coven for 3 daysand weighed again; the dry weight was expressed relative to the animal's bodymass (mg uterine weight/kg BW). The difference between wet and dry weights
allowed for determination of the percentage of weight represented by tissuewater. Determinations of the epithelial cell height and tissue wet weights wereperformed in a blinded fashion to avoid technician bias.
Results were subjected to ANOVA and differences between individual
treatment means were tested with Fisher's PLSD. These analyses were carriedout with the aid of the Statview statistics program (version 4.02; Abacus
Concepts, Berkeley, CA).
RESULTS
Estrogens increase uterine weights in ovariectomized rodents byincreasing water imbibition (edema), increasing cell size and proteinsynthesis (hypertrophy), and inducing cellular proliferation (hyperplasia). Both f3-HCH and o,p'-DDT act like estrogens in this regardexcept that the effect is only a fraction of that produced by the naturalestrogen E2. The uterine dry weight, a crude measure of the hypertrophic and hyperplastic responses, was increased when either o,p'DDT or f3-HCH was administered at 100 @g/gBW to adult ovariectomized mice for 3 days (Fig. lA); in a preliminary study, we foundthat 10 ;A.g/gBW of either compound was not sufficient (data notshown).
Animals that had been loaded with E2, @3-HCH,or o,p'-DDT wereleft untreated for 2 weeks and then one group was fasted for 2 daysand another group left on feed ad libitum. Fasted animals weighed4. 1 g less than the fed animals (25.9 ±1.89 g versus 30.0 ±2.82 g)and, although not quantified, there was visibly less i.p. fat in the fastedanimals. There were no differences in body weights due to loadingtreatments. Fasting resulted in growth of the uterus of animals that had
been loaded with @-HCH(Fig. lB). The degree of growth inductionabove control (vehicle) was nearly equivalent following the initialf3-HCH treatment (Fig. IA), and following the 2-day fast in f3-HCHloaded animals (Fig. lB), an increase of approximately 60 mg/horn/kgBW and 40 mg/horn/kg BW, respectively, was apparent. Fasting alsoincreased the degree of water imbibition in the uterus of f3-HCHloaded animals (percentage of water/uterine tissue: 79.7 ±1.49 forf3-HCH-loaded, fasted versus 73.1 ± I .33 for control, fasted,P < 0.05). There were no such effects from fasting on uterine dryweight or the proportion of water in tissues of o,p'-DDT- or E2-loadedanimals.
The hypertrophic response to estrogen is reflected in the height ofthe uterine luminal epithelial cells, and this measurement proves to bea very sensitive indicator of estrogenic stimulation (25—27).As expected, E2 dramatically increased the size of the epithelial cells in theuterine lumen; f3-HCH and o,p'-DDT also produced significant increases in the cell height, albeit a fraction of the effect of the naturalestrogen (Fig. 14). In fed, f3-HCH-loaded animals, the epithelium wasstill above control levels, indicating that there was a continued lowlevel of stimulation in these animals 2 weeks after the last injection(Fig. 2B). Fasting of the @-HCH-loaded animals induced a furtherincrease in cell height (Figs. 2B and 3); the degree of this fast-inducedresponse was the same as that seen immediately after 3 days oftreatment with f3-HCH (Fig. 2).
DISCUSSION
The results of these studies confirm the uterotrophic effects ofo,p'-DDT and f3-HCH (23, 24). They also indicate that fat stores of@3-HCHbut not of o,p'-DDT can be mobilized in a biologicallysignificant manner during fast-induced lipolysis. Earlier studies hadshown that fat stores of organochlorine compounds such as DDT,DDE, HCH, or dieldrin are mobilized during periods of dietaryrestriction (19, 28—31). In one study, insecticide residues releasedfrom adipose tissue caused nerve damage (28). In another study,dietary restriction that resulted in a 50% decrease in total body fatreduced the total fat content of HCH and dieldrin but not DDT orDDE, the concentration of the latter two compounds doubled in the fatthat remained (31). Such results indicate that xenobiotics can bedifferentially mobilized during lipolysis, some being released in a way
A B
245
TFig. 1. Uterine weights following loading treat225ments
and after fasting of loaded animals. Ovariectomized mice were treated with three daily in205jections
of vehicle (veh), 5 ng/g BW E2, 100 p@g/gBW 13-HCH, or 100 @sg/gBW o,p'-DDT (DD7).Groups of animals were killed at 24 h after the lastinjection (A). Other groups of animals were left,@ rQi85msuntreated
for 2 weeks and then either fed ad libitumor fasted for 48 h before killing (B). At the time ofsacrifice, one uterine hom was weighed, dried, andreweighed. The dry weight of the hom is expressedrelative to the BW of the animal (mg/kg BW). Data@O
B'.@125represent
means of three to four animals. Bars, SE.*,@ < 0.05 compared to the correspondingvehicle@‘105control;
**,@ < 0.05 compared to both the appropriate vehicle control and to the corresponding fedgroup..@85
that allows for redistribution in the body and others are merelyreabsorbed in the remaining fat. Although blood and tissue levels werenot measured in the present study, our results are consistent with thisnotion of differential mobilization of organochlorine compounds fromfat depots. Further testing is required to determine the extent to whichsuch differential mobilization occurs with varying body burdens ofeach compound.
There is ample evidence to suggest that obesity is a risk factor forboth breast and uterine cancers (32—37).Results of the present studysuggest a novel mechanism as the basis of this risk. It has beensuggested that the link between obesity and estrogen-responsive cancers is due to an endocrine-related mechanism such as increasednonovarian estrogen production (4, 5) or insulinemia (36). It has alsobeen suggested that the link with cancer is due to other commonaltiesbetween the two pathologies such as increased dietary fat intake (38).The present study suggests a new possibility: It may be that fat storesof xenobiotics are released in obese patients who are periodicallysubjected to dietary restrictions in an attempt to reduce their weightand that such a release of bioactive compounds may serve as a sourceof tumor promoter activity. Our results in ovariectomized mice suggest that such a hypothesis warrants further testing, including examination of the blood levels of these compounds in women before andduring a period of diet-induced weight loss. The hypothesis alsosuggests that diet-induced fat loss, not obesity per se, may be correlated to tumor etiology; thus, a woman's dieting habits may becomean important factor for determining risk of estrogen-responsivecancers.
Fig. 2. Luminal epithelial cell heights followingloading treatments and after fasting of loaded animals. The contralateral uterine hom from treatments as described for A and B in Fig. I was usedin histological examination. Tissues were fixed,paraffin embedded, and [email protected] were stainedwith H&E. The height of 75—100luminal cells wasdetermined for each animal. @,P < 0.05 comparedto the corresponding vehicle control; **, P < 0.05compared to both the appropriate vehicle controland to the corresponding fed group.
aveh E2 13-HCH DDTveh E2 g-HCH DDT
!3-HCH is one of the persistent environmental contaminants thatmay increase the risk for estrogen-responsive cancers, and releasefrom fat may be an important mechanism involved in this effect. In arecent report, j3-HCH levels in breast fat were strongly linked withcancer incidence; fat from cancerous breast was more likely to have ahigh level of f3-HCH than fat of noncancerous breast, with an oddsratio of more than 10:1 (13). f3-HCH is a component of the pesticidelindane that was widely used in agriculture for several years in thiscountry. Lindane is a mixture of isomers of hexachlorocyclohexane:the y-isomer being the active pesticide comprises approximately10—I8% of the total mix; f3-HCH makes up about 5—I2% of the totalmix (39). Manufacture of the technical grade lindane mixture wasapproximately 12 million pounds/year during the l960s when it wasused extensively as a farm pesticide (39). Although lindane is stillused as medication for head and body lice, the medication is cornposed of purified ‘y-HCH;of course, the manufacture of y-HCH stillrequires production of the other isomers as by-products. Like otherorganochlorine compounds, the isomers of HCH have accumulated inthe environment. As recently as 1992, lindane residues were found inthe waters of Resolute Bay, Northwest Territories, Canada; the concentrations of HCH had changed little since last tested in 1986 (40).@3-HCHhas been measured in fish ( I7, 18), the meat of game animals(16), and blood, fat, and milk ofhumans (21, 41—43).In fact, @-HCHbioaccumulates at a rate higher than the other HCH isomers (43, 44).Thus, @3-HCHis present in the food chain, is measurable in human fat,and is linked to the incidence of estrogen-responsive cancer. We have
1io@m
Fig. 3. Uterine histology of fed and fasted f3-HCH-loaded .mice. Shown are representative views of histological sectionsused to generate cell height measurements depicted in Fig. 2. I@Tissues were derived from (3-HCH-Ioadcd animals that were I Ieither fed (A) or fasted (B). Bars, 10 p@m.
now shown that /3-HCH that is stored in fat can be released tostimulate estrogen-responsive tissues.
@3-HCHcan be considered an xenoestrogen but its mechanism ofaction does not conform to that of other estrogenic compounds. Theterm xenoestrogen is operationally defined, i.e. , it describes an unnatural compound that produces an estrogen-like effect in a bioassaysystem such as the induction of growth of the uterus in the ovariectomized mouse. Like o,p'-DDT, @-HCHexhibits weak estrogen-likeactivity in the mouse uterus, and it increases the concentration ofprogesterone receptors in the human breast cancer cell line MCF-7(45). Other xenobiotics that exhibit estrogen-like activity in bioassaysystems interact directly with the ER or they are metabolized to activeER ligands competing with E2 in ER-binding assays (46—51). Inanother study (52), we show that j3-HCH stimulates growth of humanbreast cancer cells grown in culture, and this effect can be blocked bycotreatment with antiestrogen. Yet @-HCHdoes not compete with E2for the ER (45, 52). A lack of an appreciable increase in nuclear ERduring stimulation by @3-HCH(52) indicates that there is no metabolicconversion of the compound to an active ER ligand. These resultssuggest two things: /3-HCH may mimic estrogen action and therebyact as a promoter in the formation of estrogen-responsive tumors, andthe estrogen-like activity of /3-HCH is not mediated by the classicligand-activated ER pathway. Thus, @3-HCHmay represent a newcategory of xenobiotic capable of promoting uterine cancer.
Epidemiological studies examining a potential link between exposureto organochlorine compounds and development of estrogen-responsivetumors have yielded controversial results (53—55).Levels of DDT havebeen reported to be higher in leiomyomas, a benign uterine tumor, thanin nontumorous tissue (56); there have been no reported epidemiologicalstudies of endometrial cancer and estrogenic xenobiotics. Analysis ofserum levels of DDT showed linkage (10) or a lack of linkage (57) tobreast cancer. Tissue levels of /3-HCH but not DDT were higher in fatsurrounding cancerous breasts than in fat of noncancerous breasts (13).Such epidemiological analyses based on measurement oftissue and bloodlevels may never adequately describe the risk from these organochlorinemolecules. Numerous studies confirm that people from all parts of theworld have these compounds in their fat and blood (58). With such aubiquitous background level, it is not possible to compare exposed andunexposed cohorts of patients. In addition, the results of the present studysuggest that bioavailability of these compounds may be regulated by theintegrity of the fat stores, and individual compounds may be affecteddifferently during diet-induced lipolysis, further confounding attempts atcorrelative analysis using blood and tissue levels.
Mobilization of the weak xenoestrogen, (3-HCH, from fat depotscaused uterotrophic responses in ovariectomized mice. Such a findingsuggests a new mechanism underlying the well-established link between obesity and estrogen-sensitive cancers of the breast and uterus.Further testing will be required to determine whether such a mechanism is at work in women.
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