ICI 182,780 antagonizes the effects of estradisl on
`estrous behavior and energy balance in Syrian hamsters
`
`GEORGE N. WADE, J. BRADLEY POWERS.
`JEFFREY D. BLAUSTEIN, AND DEBORAH E. GREEN
`Department of Psychology and Neurossience and Behavior Program,
`University of Massachusetts, Amherst, llrfassachusstts 01003
`
`Wade, George N., J. Bradley Powers, Jeffrey D.
`Blaustein, and Deborah E. Green. ICI 182,780 antagonizes
`the effects of estradinl an estrous behavior and energy balance
`in Syrian han1z:bers.Am. J. Physiol. 265 (flegulamry Integrative
`Comp. Physicll. 34): R1399—R1-403, 1993.~—Tl;u-ee experiments
`examined the effects of H31 182,780, a steroidal “pure” aI1ties-
`tmgen that is thought to be active peripherally but mm. in the
`brain when given systemically, on energy balance, eslmus be-
`havior, and in viva cell nuclear binding of Pfllestrsdiol in Syr-
`ian hamsters. Pretreatment with lICl 182,780 reduced in Viva
`uptake sf [slilestradiol in uterus but not in pooled hypothala-
`mus-preoptic area. Qvariectomised Syrian hamsters were
`treated with estradiol benzoate {E33, 5 pzgjday}, ICI 182.780 (250
`pg/daylp or both EB and ICI 182380 for 4 wk. Estradicll treat-
`ment. caused significant decreases in feed intake, body weight
`and fat content, and linear gmwth. Given altme, ICI 182.’:'é:‘~0
`had no effect on these measures. When they were given concur-
`rently, ICI 182,780 attenuated the effects of estradiol an bad};
`weight, growth, and fat cnntent but not on food intake. ‘Treat-
`ment. with ICI 182,780 significantly diminished estrous behav-
`ior induced with either EB plus progesterone or with EB alcme.
`These findings support the hypothesis that. in addition to its
`actions in the brain, esnradlul acts peripherally tn modulate
`estmua luehaviur and energy balance.
`body weight; body cempositiuu; uterus; food intake; estrogen
`receptors
`
`affect other social behaviors (21), but the possibility of
`peripheral sites of action has not been tested. Second, it
`has been suggested that estradiol can act peripherally to
`alter lipid metabolism and energy balance in hamsters
`(3), but the evidence for this nutiun is rather indirect.
`Third, hamsters and rats differ in some of their re-
`sponses to antiestmgens. The older nonsteroidal amiss-
`trogens such as MER-25. (31-6223. and tamoxifen inhibit
`steroi<l—inducecl estrous behavior in both species (13, 14,
`17, 25). However, for regulation of energy balance these
`compounds are full estmgen agonists in rats, whereas
`they act as antagenists in hamsters (24.-, 25}. Thus the
`newer, steroidal antiestrogens such as 1131 182380 may
`have different effects in rats and hamsters.
`
`METHODS
`
`Animals and Housing
`
`Adult, female Syrian hamsters (Mes0crz?cetus auratus; initial
`body wt 90-110 g) of the Lak:LVG strain were obtained from
`Charles River Breeding Laboratories (Wilmington, MA). Ham-
`sters were housed in wire-bottom stainless steel cages (17.5 X
`17.5 X 25 cm) and given tap water and Purina Laboratory
`Rodent Chow (no. 5001} ad lihitum. Faod pellets were placed on
`the cage floor. A 14:10-h lightwdark cycle was maintained (lights
`an at W00 11}, and room temperature was kept at 22 i 2° C. After
`1 W35 of a-Eclimafiem to the laboratary. animals were ::wariez:£o—
`mixed via bilateral flank incisions under pezlwbarbital sudium
`anesthesia {S0 mgfkg; Sigma Chemical, St. Lcmis. MO}.
`
`Prncsdures
`
`IT IS CLEAR ‘PHAT omnlm steroids can act directly in
`the brain to affect a wide variety of behaviors and physi-
`cslogicsl functions, including social behaviors, regulatory
`behaviars, and energy balance (2, 4, 21, 23, 26}. For
`example, appropriately placed inttacerebral implants of
`estrsdicl facilitate estrous hehavicr, decrease focad in-
`In also binding of Pfljestradial. Three weeks after mariac-
`take, or stimulate valuntary exercise in ovariectomized
`tomy, hamsters were given three daily injections of sesame oil
`in = 6) or 250 ,ug ICI 182,780 in = 6). One hour after the third
`rats (6, 18, 27}; lesions of these neural lflci prevent; the
`injection, animals were injected intraperimneally with 60 ,uCi
`respective behavioral changes in response to systemic
`[“H]sstradi::nl (sp act 103 Ci/mmnl, New England Nuclear, Bos-
`estradiol treatment (7. 9, 12).
`mn, MA). One hour after injection of [3H}estradiol, hamsters
`In addition to these central actions, several lines of
`were anesthetized with penmbarbital sodium (50 mg); a blood
`evidence support the hypothesis that estradiul can act
`sample was taken via cardiac puncture with a heparinized sy-
`on nsnneursl peripheral tissues to affect. behaviors and
`ringe and then centrifuged. Hamsters were then perfused with
`energy balance in rats (22, 23, 26}. The fact that the
`cvsld saline (0.15 M). The hypothalamus-preoptic area and
`antiastrngen ICI 182,780 attenuates the effects of estra~
`uterus were rapidly disseated. Tissues were hamageniwsd, and a
`dial on energy balance and estrous behavior in ovarian-
`cell nuclear fraction was purified by a modification (8) of the
`tumized rats is consistent with the existence of periph-
`method of Zigmond and Mc:Ewen €30}. Radioactivity was ex—
`tracted from the purified cell nuclei with 3 :4 fl ml toluenehased
`eral sites of action (22). ICI182,?80 differs from other
`scintillation fluid. A. 100—;ll aliquot of plasma was transferred to
`antiestmgens in that it is highly potent peripherally but
`a scintillatisn vial containing 12 ml of scimillatimz fluid and
`it does not appear ts be active in the brain when it is
`shalien xrigcrausly. Radioactivity was cuuntsd at an efficiency of
`administered systemically (22, 28, 29).
`~45%, and counts were corrected for quenching by autnmatic
`The idea that astradiol acts hath centrally and periph-s
`external standardization. Protein in cell nuclear samples was
`erally to affect behaviors and energy balance has not
`precipitated with ethanol. dissolved in 0.3 N KOH, and esti-
`been explored in species other than rats. The present
`mated by the method cf Bradford (5). Tissue cell lmclear con-
`experiments use ICI 182,780 to investigate this possibib
`centrations of radioactivity are expressed as tissue to plasma
`ity in Syrian hamsters. Hamsters are of interest for
`Ifltlflfi (i.e., disintegrations per minutes past milligram tissue pro-
`several reasons. First, it is known that estradiol can act
`tein divided by disintegrations per minute per micraliter blood
`directly in the brain to facilitate sexual receptivity and
`plasma).
`0363-6119,f93 $2.00 Copyright '33 L998 the American Physiological Snclety
`
`R1399
`
`Astrazeneca Ex. 2109 p. 1
`Mylan Pharms. Inc. V. Astrazeneca AB IPR2016-01325
`
`

`

`R1400
`
`ANTIESTRDGEN EFFECTS IN HAMSTERS
`
`Uterus
`
`Brain
`
`A OG
`
`03 OO
`
`NOC)
`
`E2’:2
`
`UEt
`
`o
`.9CL\~.
`in
`1
`.9I-
`
`3V
`
`
`
`Energy balance. Three weeks after ovariectomy, baseline food
`intake [pouching and spillage accounted for) and body Weight
`were measured to the nearest 0.1 g. After 1 wk of data collection,
`animals were divided into four groups matched for baseline food
`intake and body weight. The groups were given daily subcuta-
`neous injections of 0.1 ml sesame oil vehicle containing 2.5%
`ethanol (n = 11), 5 pg estradiol benzoate (EB, n = 10), 250 pg
`ICI182,780 (n. = 11), or 5 pg EB plus 250 fig’ ICI 182,780 in =
`10) for 4 wk. Body weight and food intake were measured twice
`a week. On the first and last. days of injections, hamsters were
`anesthetized with pentobarbital sodium (80 mgfkg body wt),
`and naso-anal length was measured to the nearest millimeter
`with calipers while the animals were stretched with a constant
`100—g weight (24). The difference between the two lengths is an
`index of linear growth during hormone treatment.
`At the end of the experiment all animals were killed with an
`overdose of pentobarbital sodium (50 mg). Parametrial and ret-
`roperitoneal fat pads and uteri were removed and weighed. Evia-
`cerated carcasses were dried to a constant weight at 70° C, and
`carcass lipid was estimated from carcass water content. in ham-
`sters, percent carcass water and percent carcass lipid are highly
`correlated (r = 0.98; ‘Pblipid = -1.32 X %Water + 96.54; n = 289;
`Wade, unpublished data). Fat—free dry weight was calculated as
`the total eviscerated carcass weight less water and estimated
`lipid content.
`Estrous behavior. The animals that were treated with EB,
`ICI182,780, or EB plus ICI 182,780 (above) were tested for
`estrous behavior twice, once with and once without progeste-
`rone treatment. On the 3rd day of estrogen and/or antiestrogen
`treatment, all animals were given a subcutaneous injection of
`200 pg progesterone in 0.1 ml sesame oil at 0900 h and tested for
`estrous behavior 5 to 6 h later. The second test, without. pro-
`gesterone, took place on the 23rd day of estrogen and/or anti-
`estrogen treatment. For both tests, females were adapted to a
`small Plexiglas arena (30 X 36 X 30 cm) for 5 min. Sexual
`receptivity tests were begun by introducing a sexually active
`male to the arena. In addition, the females flanks and perigeni—
`tal region were continuously stimulated using an eyelid brush.
`Tests lasted for 180 s; the latency to display lordosis and the
`total time that the lordosis posture was maintained were re-
`corded. Tests were conducted 6-8 h after ligl1ts—on.
`
`Data A nalyses
`
`ICI
`Oil
`ICI
`Oil
`Fig. 1. Effects of ICI 182,780 (ICI) on in vivo uptake of [3H]estradi0l by
`cell nuclei in uterus and hypothalamus-preoptic area in ovariectomized
`hamsters. Animals were treated with sesame oil vehicle (0.1 ml) or ICI
`(250 pg) 48, 24, and 1 h before injection of [3H]estradiol. Data are
`expressed as tissue to plasma ratios, that is, disintegrations per minute
`per milligram cell nuclear protein divided by disintegrations per minute
`per microliter plasma. * P <: 0.05 vs oil—treated group.
`
`Food intake (g/day)
`
`n‘
`l4 ‘
`
`_,
`x
`'
`\_\_"'*l’)—
`on
`‘l9:'9*‘—
`..—;§<1Ci
`‘'\‘\E‘‘‘'’‘‘“.8:3(‘‘''‘‘‘~T%:~.
`‘\\-~~../.::..*2~B
`EB-a-lCl
`‘’
`
`41
`
`I
`
`l
`
`|
`
`Body weight change lg)
`
`20
`
`
`
`Data were analyzed by t tests and by one- or two -way analyses
`of variance followed by Newman-Keuls post hoc tests where
`appropriate. Differences were considered statistically signifi
`cant when P < 0.05 for two tails. Data are expressed as means
`i SE.
`
`Days oftreoimeni
`Fig. 2. Body weight gain and food intake of ovariectornized hamsters
`treated with sesame oil vehicle (0.1 ml), estradiol benzoate (EB, 5 _ug{
`clay), ICI (250 pg/day), or EB plus ICI for 4 wk. (Initial mean group body
`weights 13-4.2—135.9 g.)
`
`RESULTS
`
`In Vivo Binding of [3H]Estradiol
`Pretreatment with ICI 182,780 significantly reduced in
`vivo cell nuclear binding of [3H]estradio1 in uterus but
`not in pooled hypothalamus-preoptic area (Fig. 1), simi-
`lar to findings with rats (22). However, uterine [3H]-
`estradiol binding was reduced by only ~50% in hamsters
`compared with ~90% in rats (22).
`
`Energy Balance
`EB treatment significantly reduced food intake and
`body weight gain (Fig. 2). ICI 182,780, given alone, had
`no effect on body weight, but it significantly attenuated
`the weight—reducing actions of EB. Given alone or in
`combination with EB, ICI 182,780 had no effect on food
`intake (Fig. 2).
`
`Treatment with EB caused significant reductions in
`carcass water and lipid but not in fat—free dry weight (Fig.
`3). Once again, ICI 182,780 alone had no effect, but it
`antagonized the effects of EB on carcass lipid content.
`The changes in body fat content were reflected in the
`weights of individual fat pads (Fig. 4). EB also caused a
`significant decrease in linear growth (change in nasoanal
`length) that was prevented by concurrent treatment with
`ICI 182,780 (Fig. 4). Administration of ICI 182,780 alone
`did not affect growth.
`EB treatment caused a significant increase in uterine
`weight. As in other species (22, 28, 29), 1C1 182,780 had
`no uterotrophic effect, and at this dose, the antiestrogen
`partially antagonized the effect of EB on uterine weight
`(Fig. 4).
`
`Astrazeneca Ex. 2109 p. 2
`
`

`

`Carcass water (9)
`
`Carcass lipid (9)
`
`For-free dry wt. lg)
`
`Parametric! WAT (g)
`
`Reigoparit. WAT {g}
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`R1401
`
` c
`
`Oil
`
`EB (Cl EB+
`ICI
`
`Oll
`
`EB
`
`ICI EB+
`|C|
`
`b
`
`l8
`
`12
`6
`
`Oil
`
`EB ICI EB+
`|Cl
`
`Fig. 3. Terminal carcass composition of ovariectomized hamsters
`treated with sesame oil vehicle (0.1 ml), EB (5 cg/day}, ICI (250 ug/
`day), or EB plus ICI for 4 wk. Bars with different letters are signifi-
`cantly different (P < 0.05).
`
`Estrous Behavior
`
`Treatment with ICI 182,780, given alone or in combi-
`nation with progesterone, did not induce any signs of
`sexual receptivity in ovariectomized hamsters (data not
`shown). However, ICI 182,780 did inhibit estrous behav-
`ior induced by treatment with either EB plus progeste-
`rone or EB alone. ICI 182.780 significantly increased
`lordosis latency and decreased lordosis duration in ham-
`sters treated with EB for 2 days followed by progesterone
`(Fig. 5, top). In hamsters given EB for 23 days, concur-
`rent
`treatment With ICI 182,780
`significantly de-
`creased lordosis duration but did not affect latency (Fig.
`5, bottom).
`
`DISCUSSION
`
`These findings confirm a number of the unusual prop-
`erties of ICI 182,780. As in other species (22, 28, 29),
`ICI 182,780 appears to be a pure antiestrogen in Syrian
`hamsters. It at least partially antagonized the actions of
`estradiol on body weight, body composition,
`linear
`growth, estrous behavior, and uterine weight without
`having any agonistic (estrogenic) effects when given by
`itself to ovariectomized animals. The present results also
`support the conjecture that ICI 182,780 does not act di-
`rectly in the brain. As in rats (22), pretreatment with
`radioinert ICI 182,780, significantly reduced in vivo cell
`nuclear binding of [31-I]estradiol in uterus but not in
`pooled hypothalamus-preoptic area. Taken together,
`these two findings suggest that ICI 182,780 can he used as
`an experimental tool to dissociate central and peripheral
`actions of estradiol.
`The fact that ICI 182,780 attenuated the effects of es-
`tradiol treatment on body weight and fat content is con-
`sistent With our assertion (23, 26) that ovarian steroids
`act both centrally and peripherally to affect energy bal~
`ance. Several lines of work support the idea of distinct
`central and peripheral sites of estrogen action on energy
`balance in rats (22, 23, 26), but this appears to be the first
`evidence for this possibility in hamsters.
`The fact that treatment with ICI 182,780 completely
`blocked the effects of estradiol on linear growth (change
`in naso-anal length) may indicate that estrogen effects on
`growth are predominantly clue to nonneural actions of the
`steroid in hamsters. On the other hand, treatment with
`ICI 182,780 did not attenuate the suppressive effects of
`estradiol on food intake in hamsters. Thus it is likely that
`estradiol action in the brain is sufficient to decrease food
`
`1.00
`
`.50
`
`.25
`
`on
`
`EB
`
`ICI on
`1::
`
`on
`
`EB
`
`lC| EB+
`no:
`
`Growth (mm)
`
`o
`
`G
`
`G
`
`b
`
`B
`o
`
`4
`
`2
`
`.500
`.375
`
`.230
`
`.125
`
`"on re
`
`ICI ref
`no
`
`Uterus (g)
`b
`
`
`
`on
`
`re
`
`1:: 53+
`no
`
`Fig. 4. Terminal Weights of parametrial and retroperitoneal white adi-
`pose tissue (WAT) and uterus and change in naso-anal length (growth)
`of ovariectomized hamsters treated with sesame oil vehicle (0.1 ml), EB
`(5 pg/day), ICI (250 pg/day), or EB plus ICI for 4 wk. Ears with differ-
`ent letters are significantly different (P < 0.05).
`
`Es+rodio|+progesterone:
`it
`Latency (sac)
`Duration (sec)
`
`60
`
`40
`
`20
`
`"‘
`
`120
`
`80
`
`40
`
`EB
`
`EB+
`ICI
`
`EB
`
`EB+
`ICI
`
`Estradiol alone:
`Latency [sec]
`
`60
`
`40
`
`20
`
`Duration (sec)
`
`
`
`so
`
`40
`
`EB
`
`EB
`
`EB+
`EB+
`ICI
`ICI
`Fig. 5. Effects of ICI on estrous behavior in ovariectomized hamsters.
`Top: animals were injected with EB (5 ng) or EB plus ICI (250 Hg) for
`2 days followed by 200 pg progesterone on 3rd day, 4-5 h before testing.
`Bottom: animals were injected with EB or EB plus ICI for 23 days before
`testing. * P -< 0.05 vs. EB-treated group.
`
`intake and that no peripheral actions are required. How-
`ever, we cannot exclude the possibility that a higher dose
`of ICI 182,780 than that used in this study would be
`sufficient to antagonize the effects of estradiol on food
`intake in hamsters.
`
`Astrazeneca Ex. 2109 p. 3
`
`

`

`R1402
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`These findings in hamsters contrast with those in rate
`where ICI 182,780 treatment actually potentiated the es-
`tradiol-induced decreases in food intake (22). We sug-
`gested that in rats this action of ICI 182,780 could be due
`to the induction of a “pharmacological hysterectomy,”
`because surgical hysterectomy enhances behavioral re-
`sponsiveness to estradiol in this species (1, 20). In con-
`trast, surgical hysterectomy does not potentiate estradiol-
`induced sexual receptivity in hamsters (19),
`just as
`ICI 182,780 treatment does not potentiate the estradiol-
`induced decrease in food intake. Therefore, the difference
`between rats and hamsters in the effects of ICI 182,780
`on food intake may be a reflection of the species differ-
`ence in the actions of the uterus on behavioral respon-
`siveness to estradiol.
`
`ICI 182,780 attenuated the induction of estrous behav-
`ior, either by sequential treatments with estradiol and
`progesterone or by prolonged (23 days) treatment with
`estradiol alone. Thus, in hamsters, as in rats (22), estra-
`diol appears to act peripherally, as well as centrally(2, 4,
`21). to facilitate estrous behavior. We have suggested
`that in rats, ICI 182,780 could attenuate sexual receptiv-
`ity at least in part by blocking the effects of estradiol
`on peripheral sensory fields that are important for es-
`trous responsiveness (10, 11). Similar work examining
`the effects of ovarian steroids on peripheral sensory
`fields has not yet been done in hamsters, but it is known
`that
`sornatosensory cues
`from the perineal
`region
`play a significant role in hamster copulatory behavior
`(15, 16}.
`are possible,
`comparisons
`that
`extent
`To the
`ICI 182,780 may be more effective at inhibiting estrous
`behavior in hamsters than in rats (22). If this is in fact
`the case, the uterus might play a role in this species dif-
`ference, because surgical hysterectomy potentiates the
`effects of estradiol on sexual receptivity in rats but not
`in hamsters (1, 19). Thus in rats ICI 182,780 could con-
`currently inhibit estrous behavior
`(perhaps by inhi-
`biting an action of estradiol on peripheral sensory
`fields) and facilitate estrous behavior (via a pharmaco-
`logical hysterectomy facilitating estrogen action in
`the brain). According to this hypothesis, only the inhibi-
`Lory actions would be evident in hamsters.
`It appears as though the dose of ICI 182,780 we have
`used (250 pg/day) is more effective in reducing uterine
`weight and in vivo cell nuclear uptake of l3H]estradiol in
`rats than in hamsters (22). At this time it is not clear
`whether this effect is due to 1) species differences in
`ICl182,780 absorption, delivery, or clearance; 2) dif-
`ferences in affinity for estrogen receptors; or 3) some
`other factor(s). Differences in the EB doses that were
`used (2 pg in rats, 5 ug in hamsters) could explain the
`differences in the inhibition of uterine weight but can-
`not account for the differences in inhibition of [3H]es-
`tradiol binding. Whatever the basis for this species dif-
`ference in ICI 182,780 potency in the uterus, the impor-
`tant point remains that
`in both rats and hamsters
`treatment with ICI 182,780 attenuates the effects of es-
`tradiol on energy balance and on estrous behavior with-
`out interfering with neural estrogen binding. Therefore,
`these results support the hypothesis that estradiol affects
`
`energy balance and estrous behavior via both neural and
`nonneural sites of action.
`
`We are grateful to Jay Alexander, Robin Lempicki, and Joanne Tur-
`cotte for their expert technical assistance and to Alan E. Wakeling of
`Zeneca (ICI) Pharmaceuticals for the gift of ICI 182,780.
`This work was supported by Research Grants N340873. DK-32976,
`and NS-19327, by Research Scientist Award MH-00321, and by Re-
`search Scientist Development Award MH-00885 from the National 111-
`stitutes of Health.
`Address reprint requests to G. N. “fade, Dept. of Psychology, Univ.
`of Massachusetts, Amherst, MA 01003.
`Received 8 March 1993; accepted in final form 5 May 1993.
`
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`
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