[CANCER RESEARCH 41. 4432-4436, November 1981]

Melatonin Inhibition and Pinealectomy Enhancement of 7,12-Dimethylbenz(a )anthracene-induced Mammary Tumors in the Rat

Lawrence Tamarkin, Michael Cohen, David Roselle, Cheryl Reichert, Marc Lippman, and Bruce Chabner

Intramural Research Program, National Institute of Child Health and Human Development ¡L.T.], and Division of Cancer Treatment [M. C., D. R. M. L., B. C.], andLaboratory of Pathology 1C P.], National Cancer Institute. NIH, Bethesda. Maryland 20205

ABSTRACT

The effects of the pineal hormone, melatonin, and of pineal-

ectomy on the incidence of mammary adenocarcinoma inSprague-Dawley rats treated with 7,12-dimethylbenz(a)-

anthracene (DMBA) were investigated. Melatonin (2.5 mg/kg),begun on the same day as DMBA (15 mg) treatment and givendaily in the afternoon for 90 days, significantly reduced theincidence of mammary tumors from 79% (control) to 20%(treated) (p < 0.002). Rats pinealectomized at 20 days of ageand treated with 7 mg of DMBA at 50 days of age had a higherincidence of tumors (88%) compared to control animals (22%).Fifteen mg of DMBA, which resulted in a higher incidence oftumors, reduced the difference between pinealectomized andcontrol animals. Melatonin only partially reversed the effects ofpinealectomy, reducing the incidence from 87% (pinealectomyalone) to 63% (pinealectomy plus melatonin); however, thetumor incidence was still lower (27%) in nonpinealectomized,melatonin-treated animals. Assessment of plasma prolactin,luteinizing hormone, follicle-stimulating hormone, estradiol,and cortisol in DMBA-treated tumor-free and tumor-bearing

animals revealed a significantly lower plasma prolactin concentration [27 ±5 (S.E.) ng/ml] in melatonin-treated animals ascompared to vehicle-treated animals [65 ± 8 ng/ml]. Theconcentration of plasma prolactin was less in melatonin-

treated, pinealectomized rats (55 ±10 ng/ml) as compared tovehicle-treated, pinealectomized animals (101 ± 13 ng/ml).

Other hormones were not affected by melatonin treatment.These data support the hypothesis that melatonin inhibits thedevelopment of DMBA-induced mammary tumors in the rat

while removal of the pineal gland stimulates development ofsuch tumors. Additionally, these experiments provide evidencethat these effects may be mediated by a suppression of plasmaprolactin levels.

INTRODUCTION

Rat mammary tumors induced by DMBA1 respond to a num

ber of hormone manipulations (3, 8). DMBA-induced tumors

are often prolactin dependent; experimental manipulations thatreduce circulating levels of prolactin, such as hypophysectomyor treatment with 2-bromocryptine, inhibit tumor induction by

DMBA (24, 25). Conversely, transplantation of a pituitary orwithdrawal of 2-bromocryptine promotes tumor growth (24,

25).Ovarian steroids also influence tumorigenesis following

DMBA administration. For the initiation of mammary tumors,the presence of estrogens may be required (3, 4, 8). However,

' The abbreviations used are: DMBA, 7,12-dimethylbenz(a)anthracene; LH,luteinizing hormone: FSH, follicle-stimulating hormone; i.g.. intragastric.

once induced, most mammary tumors can grow in the absenceof ovarian steroids if prolactin levels are elevated; estrogenreplacement by itself is usually insufficient to maintain tumorgrowth in hypophysectomized-ovariectomized DMBA-treatedrats (3, 18). These observations suggest a permissive role forestrogens and an active role for prolactin in the promotion ofDMBA mammary tumors.

Another line of investigation has suggested an inhibitoryeffect of the pineal gland on reproductive function and prolactinsecretion. In the Syrian hamster, the pineal gland and itshormone melatonin induce gonadal collapse (7, 14, 16, 17,19, 21, 22). Long-term daily afternoon injections of melatonincan induce anovulation that essentially mimics the pineal-me

diated response to a lighting schedule that inhibits reproductivefunction (19). The pituitary hormone affected most rapidly bymelatonin treatment is prolactin, which is markedly suppressedin the circulation (14, 16).

In the rat, long-term daily afternoon injections do not induce

a change in reproductive function (5). However, removal of theolfactory bulbs may change the sensitivity of rats to exogenousmelatonin, since anosmic female rats treated daily with melatonin have lowered levels of pituitary prolactin (15). The possible inhibitory role of the pineal gland and melatonin on reproductive function and prolactin secretion led to this series ofexperiments which test the effects of the pineal gland and itshormone melatonin on induction of mammary tumors in the ratby DMBA.

MATERIALS AND METHODS

Animals. Sprague-Dawley rats were supplied by Zivic-Miller Co.,

Allison Park, Pa., and were housed in windowless rooms maintainedon a lighting schedule of 12 hr of light per day (lights on at 6 a.m. forExperiments 1 and 2; lights on at 4 a.m. for Experiment 3). All animalswere allowed access to food and water ad libitum.

Surgery. Pinealectomies were performed at 20 days of age according to the procedure of Hoffman and Reiter (6). At 2 weeks followingsurgery, the animals were shipped to our laboratories.

Radioimmunoassays. Radioimmunoassays for estradiol, cortisol,prolactin, LH, and FSH were performed on plasma samples collectedfrom animals used in Experiment 3. The reagents for the prolactin, LH,and FSH radioimmunoassays were provided by the National Institute ofArthritis, Metabolism, and Digestive Diseases. Plasma prolactin isreported in ng of prolactin Preparation RP-1, using rat prolactin antibody S-7. Plasma LH is reported in ng of LH Preparation RP-1, usingrat LH Antibody S-4. Plasma FSH is reported in ng of FSH PreparationRP-1, using rat FSH Antibody S-9.

Statistics. Data from Experiments 1 and 2 were analyzed by thegeneralized Wilcoxon test. For Experiment 3, the overall equality of the5 test groups was compared using the generalized Kruskal-Wallis test.

Statistical differences of body weights and hormone levels in Experiment 3 were determined by Student's f test.

Experiment 1: Effect of Melatonin on the Incidence of DMBA-

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Melatonin, the Pineal Gland, and DMBA Tumors

induced Mammary Tumors. Sixty 50-day-old rats were given 15 mgof DMBA in peanut oil by i.g. intubation. On the same day, 30 of theseanimals received 500 fig melatonin (Sigma Chemical Co., St. Louis,Mo.) i.p. The remaining 30 animals received 0.25 ml vehicle (4%ethanol in phosphate-buffered saline). The melatonin and vehicle injec

tions were administered at 4 p.m. and were continued daily for 90consecutive days. Beginning 1 week following DMBA treatment andfor 140 days, the animals were palpated twice weekly, and the incidence of tumors was recorded. Tumors were measured with verniercalipers, and when the tumors reached 2.5 cm in single diameter the

animals were sacrificed.Experiment 2: Effect of Pinealectomy on the Incidence of DMBA-

induced Mammary Tumors. At 20 days of age, 36 rats underwentpinealectomy and 36 animals had a sham pinealectomy. When the ratswere 50 days old, 7 mg of DMBA were administered to all rats by i.g.intubation. After 1 week, the surviving animals were examined twiceweekly for tumors. The study was terminated 240 days following theadministration of DMBA, and each animal was checked for successful

pinealectomy at autopsy.At 20 days of age, 30 rats were pinealectomized and 30 rats were

sham pinealectomized. All animals were given 10 mg of DMBA at 50days of age. One week following DMBA treatment, the presence oftumors was recorded in the remaining animals, and, as previouslydescribed, animals with tumors 2.5 cm in diameter were sacrificed,and the absence of the pineal gland was determined. The study wasterminated 240 days following the administration of DMBA.

Experiment 3: Effect of Melatonin and/or Pinealectomy on theIncidence of DMBA-induced Mammary Tumors. When animals were52 days old, 15 mg of DMBA were administered to 175 rats by i.g.intubation, and animals that died within 1 week following DMBA treatment were not included in the experiment. Seventy of the remaininganimals had a pinealectomy at 20 days of age, and one-half of these

received daily injections of 500 ¡igmelatonin between 11 a.m. and 12noon, beginning 1 day after DMBA treatment. The remaining 35 pinealectomized animals were given injections of 0.25 ml vehicle (4%ethanol in phosphate-buffered saline) at the same time of day. Thirty-

five animals that underwent a sham operation at 20 days of age werealso given injections of 0.25 ml vehicle daily at this same time of day.Seventy intact animals were divided into 2 groups: one receiving dailyinjections of 500 ng of melatonin between 11 a.m. and 12 noon; andthe other receiving daily injections of 0.25 ml of vehicle at this sametime of day. Treatment began 1 day after DMBA injection and wascontinued for 92 days. The 5 study groups were: Group 1, pinealectomy plus melatonin; Group 2, pinealectomy plus vehicle; Group 3,sham pinealectomy plus vehicle; Group 4, intact plus melatonin; Group5, intact plus vehicle.

During the course of this experiment, animals tnat developed mammary tumors >1 cm in diameter were sacrificed at weekly intervals at10 a.m. Tumor-free animals were sacrificed 92 days after DMBA

treatment at the same time of day. Each animal was sacrificed bydecapitation, and tumors were removed and stored in 10% formalinuntil processed for histological evaluation. Trunk blood was collectedin heparinized tubes and centrifuged, and the plasma was harvestedand stored at -20° until assayed by radioimmunoassay for prolactin,

LH, FSH, estradici, and cortisol.

RESULTS

Inhibition of Mammary Tumors by Melatonin in DMBA-treated Rats. During the first 90 days following a 15-mg doseof DMBA, 50% of the vehicle-injected rats developed tumors,while none of the melatonin-injected animals had tumors (Chart

1). Melatonin injections were discontinued on Day 90 followingDMBA. By Day 140 (50 days after discontinuation of melatonin), 79% of the vehicle-injected animals had developedtumors, while significantly less, 20%, of the melatonin-injected

animals developed breast tumors (p < 0.002).Enhancement of Mammary Tumor Development by Pine

alectomy in DMBA-treated Rats. To induce a lower percentageof intact animals developing tumors, low doses of DMBA wereadministered. After a dose of 7 mg of DMBA, 48% of thepinealectomized animals developed tumors within 2 months,while none of the intact animals developed tumors during thissame period of time (Chart 2). By the 240th day after DMBAtreatment, 88% of the pinealectomized animals had tumors, afigure which was significantly greater than the 22% tumorincidence in intact animals (p < 0.002).

In a second study, in which 10 mg of the DMBA wereadministered, the differences between pinealectomized andcontrol animals were not as striking (Chart 3). By the 230thday following treatment, 46% of the pinealectomized animalsdeveloped tumors compared to 21% of the intact animals (p< 0.03).

A final comprehensive experiment was designed to determine the effect of pinealectomy and melatonin administrationon plasma hormone concentrations and on the incidence ofmammary tumors. A high dose of 15 mg of DMBA was administered, and by the end of the study 87% of the intact animalsdeveloped tumors (Chart 4). There was no further increase inthe incidence of tumors in the pinealectomy group. Pinealectomized animals which received daily melatonin injections had

34

M 20

I i.i12ë s

-,

•¿�Melatonin

O Control

20 40 60 80 100 120

DAYS POST-DMBA (15 mg]140

Chart 1. Incidence of breast carcinoma in Sprague-Dawley rats given 15 mgof DMBA at 50 days of age (Day 0). From 0 to 90 days, all animals received dailys.c. injections of melatonin (500 fig/animal) or vehicle (control) at 4 p.m. (lightson from 6 a.m. to 8 p.m.). All animals were examined twice weekly for tumorsuntil 140 days after DMBA treatment.

20 40 60 80 100 120 140 160 180 200 220 240

DAYS POST-DMBA (7 mg)

Chart 2. Incidence of breast carcinoma in Sprague-Dawley rats pinealectomized or sham operated at 20 days of age and given 7 mg of DMBA at 50 days ofage (Day 0). All animals were examined twice weekly for tumors until 240 daysafter DMBA treatment.

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L. Tamarkin et al.

a lower incidence of tumors (63%) than did either intact orpinealectomized animals; however, the difference was not statistically significant. Only 27% of the pineal-intact animalsreceiving melatonin had tumors by the end of the experiment,which was significantly less than that observed in the controlor pinealectomy groups (p < 0.002). Irrespective of treatmentgroup, the mean number of tumors per tumor-bearing rat was

1.4.In Experiment 3, for each animal possessing at least one

mammary tumor, a single 4-fim hematoxylin-eosin-stained sec

tion of the dominant mammary tumor nodule was examinedand classified according to standard histological criteria (28).Of the 86 tumors examined, 84 were malignant epithelial tumors, one was a fibroadenoma, and in one section no tumorwas identified. The tumors were almost exclusively papillarycarcinomas, with occasional cribriform and/or comedocarci-noma variants in portions of the tumors. Mitoses were readilyapparent, and necrosis as well as an infiltrating componentwere apparent in some of the sections. The carcinomas wereusually intersected by fibrous septae which were frequentlyinfiltrated by a mild to moderate chronic inflammatory infiltrate.The low incidence of benign fibroepithelial tumors in this studyis probably attributable to both the relatively young age of theanimals at necropsy and the selection of the dominant, rapidlygrowing mass for histological evaluation. Mammary tumorswere not analyzed histologically in Experiments 1 and 2.

The body weight of each animal in Experiment 3 was determined 5 weeks after DMBA treatment. At this point in theexperiment, there was no significant difference in body weightamong the groups. During the course of the experiment, bodyweights of the animals from the 5 groups were determined andcompared at the first appearance of tumor. No significant

20 40 60 80 100 120 140 160 180 200 220 240

DAYS POST DMBA HO mgl

Chart 3. Incidence of breast carcinoma in Sprague-Oawley rats pinealectomized or sham operated at 20 days of age and given 10 mg of DMBA at 50 daysof age (Day 0). All animals were examined twice weekly for tumors until 240 daysafter DMBA treatment.

difference was noted in the body weights of the animals fromthe 5 treatment groups.

We examined the effect of these experimental manipulationson the plasma concentrations of various pituitary and steroidalhormones known to influence tumor growth and gonadal function. No significant differences among the groups were observed in the concentrations of LH, FSH, cortisol, or estradici(Table 1). Plasma prolactin was significantly suppressed inanimals given daily injections of melatonin compared to vehicle-

injected animals (p < 0.01). Pinealectomized animals giveninjections of melatonin also had lower levels of plasma prolactinthan did pinealectomized, vehicle-treated animals (p < 0.01).

DISCUSSION

We have recently summarized epidemiológica! and anecdotalclinical evidence which supports the hypothesis that the pinealgland and its secretions may inhibit the development of breastcarcinoma in humans (2). In order to study this possible relationship in an animal model, we have investigated the effect ofthe pineal gland and melatonin in the development of DMBA-

induced tumors in the rat. The present studies provide evidencefor a modulating role of melatonin and the pineal gland in thedevelopment of breast cancer in this system. Long-term daily

administration of melatonin inhibited tumorigenesis, whereaspinealectomy increased the incidence of breast tumors. Mea-

293643505764 71 788592

DAYS POST DMBA (15 mg)

Chart 4. Percentage of tumor-free rats treated with 15 mg of DMBA at 50days of age (Day 0). Pinealectomies or sham operations were performed at 20days of age, and melatonin (500 fig/animal) or vehicle injections were administered s.c. daily from Day 1 through Day 92. All animals were examined once aweek for tumors until 92 days after DMBA treatment.

Table 1Plasma concentrations of prolactin, LH, FSH, estradici, and cortisol in intact or pinealectomized DMBA-treated rats given 250 ¡igof melatonin or vehicle daily

TreatmentMelatonin

VehiclePinealectomized-melatoninPinealectomizedSham operatedNo.

ofrats16

26231821Prolactin

(ng/ml)27± 5""

65 ± 855 ±10°

101 ±1381 ± 9LH

(ng/ml)54

± 675 ±1065 ±1071 ±1690

i 16FSH

(ng/ml)60

± 7112 ±23

99 ±1192 ±15

120 ±25Estradici

(pg/ml)29

± 432 ± 342 ± 543 ±1040 ± 6Cortisol

(/ig/dl)13

±212 ±2

9 ±112 ±112 ±2

Mean ±S.E.6 p < 0.01 compared to vehicle group.c p < 0.01 compared to pinealectomized group.

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Melatonin, the Pineal Gland, and DMBA Tumors

sûrementof various hormones suggests that these effects maybe mediated by changes in circulating levels of prolactin.Melatonin administration depressed prolactin concentrations inthe plasma, while pinealectomized rats had the highest prolactin levels and the highest tumor incidence of all groups studied.However, these experiments do not conclusively indicate thatregulation of plasma prolactin is the sole or necessary mechanism by which the pineal gland and melatonin may influencetumorigenesis following DMBA administration.

Earlier studies indicate that, contrary to inhibiting prolactinsecretion, melatonin administration actually causes an increasein circulating prolactin (14). More recent studies have demonstrated that daily afternoon melatonin treatment of anosmicfemale rats caused a reduction in serum and pituitary prolactinlevels whereas the same treatment given to intact controls hadno effect (15). Perhaps the effect of anosmia is to increase thesensitivity of the animal to melatonin, a possibility which mayalso apply to the DMBA-treated rat.

It should be noted that the paradigm for the daily injection ofmelatonin was drawn from previous studies in Syrian hamsters,in which melatonin inhibited reproductive function only whenadministered at specific times of day (21). Long-term adminis

tration of melatonin to hamsters induces testicular regressionand decreases levels of prolactin in males and induces ano-

vulation in females only if administered 1 to 4 hr prior to theonset of the dark period of the day; injections of melatonin inthe morning have no effect on reproductive function (21). Inthe present study, the animals were given injections during thisperiod of maximum melatonin sensitivity. However, we chosea 20-fold higher daily dose of melatonin to be administered tothe DMBA-treated rats to ensure a long period of elevated

levels of circulating melatonin.The pineal gland itself may influence the sensitivity of target

organs to exogenous melatonin. Melatonin is synthesized bythe pineal and secreted into the circulation in a diurnal rhythmcharacterized by low levels during the day and elevated levelsat night (9-11, 20, 26). The presence of this daily rhythm may

affect the sensitivity of specific target tissues to the hormone.Consequently, administration of melatonin prior to the nocturnal increase in endogenous melatonin may be most effectivebecause melatonin target tissues are most sensitive to thehormone at this time.

Removal of the pineal gland has been shown to abolish themelatonin rhythm in the circulation of the rat (9). In the presentstudy, pinealectomy enhanced mammary tumorigenesis inDMBA-treated rats, suggesting that the pineal gland, through

the daily melatonin rhythm, may have an inhibitory effect ontumor induction by DMBA. However, hormone replacement bya single daily injection of melatonin to pinealectomized ratspartially restored the tumor incidence to that of untreatedanimals, but not to the even lower incidence observed in intact,melatonin-treated rats.

One explanation of this observation is that removal of thepineal gland, which results in the loss of the melatonin rhythm,might cause a loss in end organ sensitivity to melatonin andmay abolish the time-of-day sensitivity to exogenous melatonin.

A single daily injection of melatonin might not be sufficient toreinitiate hormonal sensitivity. This hypothesis is supported bystudies in the hamster, in which a single daily injection ofmelatonin was not sufficient to induce gonadal collapse inpinealectomized animals; rather, 3 daily injections of melatonin

were necessary to induce gonadal collapse in pinealectomizedanimals (19, 21). A parallel study of multiple melatonin injections in DMBA-treated rats has not been performed.

Alternatively, removal of the pineal gland might result in theloss of another substance(s) in addition to melatonin that mightalso have a role in the inhibition of tumorigenesis. One hypothesis is that melatonin functions within the pineal gland to initiatethe release of other pineal products, most notably argininevasotocin, which has been shown to act on gonadal tissue (12,13). The hypothesis is supported by studies in vivo whichrevealed the uptake of [3H]melatonin by the pineal gland and

by another study demonstrating the release of arginine vasotocin into cerebrospinal fluid by melatonin (12, 27). However,arginine vasotocin has been shown to increase prolactin levelsin the rat (23), and a role for this molecule in tumor inductionhas not been shown. Alternatively, other pineal products, possibly regulated by melatonin, may play a role in DMBA-medi-

ated mammary tumors.A previous study concluded that melatonin inhibited tumor

induction in DMBA-treated rats (1). Additionally, this study

demonstrated that melatonin treatment did not reduce tumorincidence in rats whose pineal melatonin rhythm was suppressed by constant exposure to light (1 ). Thus, this previousstudy provides additional evidence that mammary tumorigenesis can be inhibited by daily melatonin treatment provided thatthe melatonin rhythm is present. These studies suggest that apossible role of pineal function and the daily melatonin rhythmshould be examined as an etiological factor in human breastcancer.

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1981;41:4432-4436. Cancer Res   Lawrence Tamarkin, Michael Cohen, David Roselle, et al.   the Rat

)anthracene-induced Mammary Tumors ina7,12-Dimethylbenz(Melatonin Inhibition and Pinealectomy Enhancement of

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