Photoperiodic Effects on Tumor Development and Immune Function

Effects on Tumor and Immune Function Photoperiodic Development Nelson and Joan M. C. Blom 2 Randy J. 1 Behavioral Department of Psychology, Neuroendocrinology Group, The Johns Hopkins University, Baltimore, Maryland 21218 Abstract Seasonal changes in adaptations associated with winter coping strategies have been frequently studied. Central among the suite of energy-saving, winter-coping strategies is the suspension of reproductive activities. The inhibition of reproduction by nontropical rodents is mediated by daylength changes. Although balanced annual energy budgets are critical, survival and subsequent reproductive success also require avoiding predators, illness, and early death. Because the stressors of winter could lead to suppressed immune function, we hypothesized that animals should have evolved survival strategies involving immunoenhancement. Short daylengths provide a predictive cue to individuals that could be used to enhance immune function in advance of stress-induced immunosuppression. In Experiment 1, adult female deer mice (Peromyscus maniculatus) were housed in either long (LD 16:8) or short (LD 8:16) days for 8 weeks, then injected with the chemical carcinogen 9,10-dimethyl-1,2benzanthracene (DMBA) dissolved in dimethyl sulfoxide (DMSO) or with the DMSO vehicle alone. Animals were evaluated weekly for 8 weeks after injection. None of the animals treated with DMSO developed tumors in any of the experiments. Nearly 90% of the long-day deer mice injected with DMBA developed squamous cell carcinoma. None of the short-day deer mice injected with DMBA developed tumors. Small lesions developed at the site of injection; short-day females had less severe lesions and healed faster than long-day females. Immunoglobulin G (IgG) response to i.p. injection of sheep red blood cells (SRBC) did not differ between photoperiodic conditions. The role of estrogens in the photoperiodic responses was evaluated in Experiment 2: Ovariectomized or sham-ovariectomized deer mice received estradiol benzoate replacement therapy or a control procedure in long daylengths for 8 weeks prior to injection of DMBA or DMSO, then were monitored for 8 additional weeks. Females treated with DMBA developed tumors at the same rate, regardless of estrogen manipulation. Estrogen did not affect healing rates. In Experiment 3, female deer mice were injected with a slurry of microspheres that either contained bromocriptine or were empty. Suppression of prolactin with bromocryptine resulted in a decrease of tumor incidence from 55.6% to 24% in long-day females 8 weeks after injection with DMBA. Healing rates were not affected by prolactin manipulations. Silastic capsules that were filled with either melatonin or cholesterol were implanted into long-day female deer mice in Experiment 4; 8 weeks later, females received an injection of either DMBA or DMSO, then were monitored for 8 weeks. Approximately 66% of females implanted with cholesterol and injected with DMSO developed histologically verified tumors. None of the melatonin-implanted mice developed tumors. Melatonin did not affect healing rates. Taken together, these results indicate that photoperiod can exert a functionally significant effect on immune processes and clinical disease. Key words immunoglobulin, seasonality, daylength, prolactin, melatonin, DMBA, tumorigenesis 1. To whom all correspondence 2. Present address: Center for mammary cancer, carcinoma, estrogen, should be addressed. Neuropharrnacology, Via Balzavett 9, 20133 Milan, Italy. 233 Seasonal changes in energy requirements have been documented for many species of nontropical rodents (Bronson, 1989; Goldman and Nelson, 1993). The energy required for thermoregulation reaches a peak during the winter, when energy availability is typically reduced. The energetic &dquo;bottleneck&dquo; created by high energy demands during low energy availability has led to the evolution of many adaptations to cope with winter (Blank, 1992; Wunder, 1992). Reproduction is energetically expensive, and the curtailment of breeding is central among the suite of winter-coping strategies among boreal and temperate zone rodents (Blank, 1992; Wunder, 1992). Successful individuals have evolved strategies to maximize the length of their breeding effort without jeopardizing their survival vis-d-vis energy usage. These temporal strategies require the ability to ascertain the time of year in order to phase energetically expensive activities to coincide with peak energy availability and other local conditions that promote survival (Bronson, 1989; Moffatt et al., 1993). Photoperiod can be used by nontropical animals as a very precise temporal cue for time of year. This temporal information is often employed by individuals to anticipate predictable seasonal environmental changes, and to initiate or terminate specific seasonal adaptations in order to maintain a positive energy balance. Maintenance of a positive energy balance is obviously required for survival, and the vast majority of studies of seasonality have focused upon the temporal organization of energetic adaptations (for reviews, see Nelson et al., 1990; Bronson and Heideman, 1994). However, other threats to survival must also be met in order for individuals to increase their fitness. They must avoid encountering predators, engaging in potentially dangerous interactions with conspecific competitors, and succumbing to disease. Certainly, energetics can interact with immune function; a negative energy balance can sufficiently weaken animals to the extent that they increase their risk of infection and death (Kelley, 1985). Other conditions perceived as stressful can suppress immune function and promote opportunistic diseases. Stressful conditions such as reduced food availability, low ambient temperatures, overcrowding, lack of cover, or increased predator pressures can recur on a seasonal basis, leading to seasonal changes in immune function among individuals, as well as seasonal changes in population-wide disease and death rates (Lee and McDonald, 1985; Lochmiller et al., 1994). Because these stressful conditions are somewhat recurrent, we have hypothesized that animals may have evolved mechanisms to combat seasonal stress-induced reductions in immune function. From an evolutionary and ecological perspective, it is reasonable to expect that rodents have evolved the ability to forecast recurrent conditions associated with immunosuppression and to maximize immune function in advance of these challenging conditions. In addition to the well-studied seasonal cycles of mating and birth, there are also seasonal cycles of illness and death among many populations of animals (e.g., Bradley et al., 1980; McDonald et al., 1981; Mihok et al., 1988; Lochmiller et al., 1994). Furthermore, seasonal fluctuations in immune function and the incidence of opportunistic diseases have been well documented in a variety of species, including humans (Christian, 1978; McDonald et al., 1981; Maestroni and Conti, 1991; Blom, 1992). For example, the numbers of circulating leukocytes in mice (Mus), rats (Rattus), rabbits (Lepus), dogs (Canis), ground squirrels (Citellus), voles (Microtus), and humans were reported to be elevated during the autumn and winter; spleen and thymus masses were also reported to be highest during autumn and winter in deer mice (Peromyscus maniculatus), prairie voles (Microtus ochrogaster), and northern red-backed voles (Clethrionomys rutilus) (for a review, 234 see Blom, 1992). These seasonal changes in immune function appear to be mediated by daylength. Previstudies have indicated that short-day exposure increases splenic masses of deer mice and Syrian hamsters (Mesocricetus auratus); short-day exposure also elevates total lymphocyte and macrophage counts (Vriend and Lauber, 1973; Brainard et al., 1985, 1988; Champney and McMurray, 1991; Blom et al., 1994). Enhancement of immune cell numbers should be particularly crucial to young bom near the end of the breeding season. Indeed, information about short daylengths is transmitted by mothers to their offspring in utero, which accelerates immune cell production in the young (Blom et al., 1994). Melatonin appears to mediate photoperiodic enhancement of immune function both in vivo and in vitro; however, some immunosuppressive effects of melatonin have also been reported (reviewed in Maestroni and Conti, 1991). Importantly, virtually all of the studies examining the interaction between melatonin and immune function have been conducted on humans or inbred strains of laboratory mice and rats-species that do not usually display robust responsiveness to melatonin or photoperiod. Also, many previous studies have not controlled for the suppressive effects of short days or melatonin treatment on reproductive function. Reproductive steroid hormones, as well as prolactin, can directly affect immune function (for reviews, see Grossman, 1984, 1985; Bemton et al., 1988; Hartmann et al., 1989). Consequently, the reported photoperiodic effects on immune function may merely reflect steroid, prolactin, or melatonin effects on immune function. Associated with its effects on immune function, melatonin has also been reported to possess oncostatic properties against several types of tumors both in vivo and in vitro (reviewed in Blask, 1984; Blask et al., 1991). In common with studies of melatonin and immune function, the majority of studies investigating the effects of melatonin on cancer are based on inbred strains of mice and rats, which are not particularly responsive to melatonin. Furthermore, previous studies have administered melatonin with little regard for the temporal characteristics of the melatonin treatment (Blask et al., 1992). Consequently, the adaptive function of this pineal hormone in mediating seasonal changes in immune function may have become obscured. The present study investigated the influence of daylength on immune function and tumorigenesis in female deer mice (Peromyscus maniculatus). We hypothesized that short days should enhance immune function and suppress tumor development in this species. The roles of estrogen, prolactin, and melatonin in tumor development were also assessed. ous METHODS ANIMALS AND HOUSING CONDITIONS Adult (>50 days of age) female deer mice (Peromyscus maniculatus bairdii) were obtained from our colony, which was originally derived from animals obtained from the Peromyscus Stock Center at the University of South Carolina. Animals were maintained with their littermates until weaning at 21 days of age (or after their body mass was >7 g), and were x x 7.5 13 at 21° ± 2°C in housed thereafter cm) (28 cages polypropylene individually and a relative humidity of 50% ± 5%. Food (Prolab 1000; Agway, Syracuse, NY) and tapwater were available ad libitum throughout all experiments. Deer mice were maintained in either long (LD 16:8; lights-on at 0700 hr Eastern Standard Time [EST]) or short (LD 8:16; lights-on at 1000 hr EST) photoperiods. 235 GENERAL EXPERIMENTAL PROCEDURES After 8 weeks in their respective photoperiodic conditions, animals were injected subcutaneously (s.c.) in the lower left abdominal region with the chemical carcinogen 9,10-dimethyl1,2-benzanthracene (DMBA) dissolved in dimethyl sulfoxide (DMSO) (50 mg/kg) or with the DMSO vehicle alone. All animals were palpated weekly to detect tumor onset and growth during an 8-week postinjection period. After 8 weeks, the animals were anesthetized with methoxyflurane (Metofane; Pitman Moore, Fort Washington, NJ) vapors and weighed, and a blood sample was obtained; animals were then killed by cervical dislocation. Tumors, spleens, and (in some experiments) uteri were dissected at autopsy and the weights recorded. Tumors were fixed for later histopathological diagnoses. Animals were examined daily and also monitored by the veterinary staff; during the course of the experiments, the injection sites were observed to develop skin irritation resulting in lesions. The severity and rate of healing of these lesions were assessed on a weekly basis in some of the experiments with a subjective 5-point scale: (0) no lesions, (1) barely detectable lesions, (2) mild lesions (<3 mm), (3) moderate lesions (3-5 mm), or (4) severe lesions (>5 mm). Animals with severe lesions or with tumors >5 mm in diameter were sacrificed on the day that these were discovered (n 3 for all experiments). = BLOOD SAMPLES Blood samples (250 ~,1) were collected between 0900 hr and 1200 hr EST from the retroorbital sinus, stored at room temperature for 1 hr, declotted, and then centrifuged at 3500 rpm for 1 hr at 4°C. Serum was separated and stored frozen at - 80°C until assayed for immunoglobulin G (IgG). IMMUNE ASSAYS AND PATHOLOGY Sheep red blood cells (SRBC) were obtained from Truslow Farms (Chestertown, MD). The SRBC (3 ml) were washed three times in 0.2 M phosphate-buffered saline (PBS; pH 7.2); they were then resuspended in PBS for a final concentration of 0.1%. IgG levels in the blood samples were determined by means of a sandwich enzymelinked immunosorbent assay (ELISA) that was developed and validated for use in deer mice in our laboratory. Serial dilutions yielded values in parallel to the standard curves. We incubated 96-well immunoplates (Nuncg, MaxiSorp) overnight at 4°C with 100 Wl/well of a goat polyclonal antibody against house mouse IgG (Cappel) diluted 1:3000 in a carbonatebicarbonate buffer (0.1M, pH 9.6). The following day the plates were washed four times with PBS (0.05 M, pH 7.4) containing 0.05% Tween-20 and 0.001% of NaN3; an automatic microplate washer (BioRad, model 1550) was used. A standard curve (upper limit, 1000 fLg/ml; lower limit, 0.001 fLg/ml) was prepared using purified mouse IgG (Sigma) diluted in standard diluent (PBS [0.05 M, pH 7.4] containing 0.05% Tween-20). The standards (100 iLl/well in triplicate), and samples of deer mouse serum (100 ~,1/well in duplicate) diluted 1:100 with standard diluent, were placed in wells on the plates. The plates were incubated overnight at 4°C. The following day the plates were washed, and 100 ~,1 of alkaline phosphatase-conjugated sheep antimouse IgG diluted 1:2500 in standard diluent were added to each well. The plates were incubated overnight at 4°C. The following day the plates were again washed, and 100 fLI of substrate buffer (0.1mMp-nitrophenyl phosphate 236 in diethanolamine buffer [0.1M, pH 9.5] containing 5 mM MgC’2) was added to each well. The plates were incubated for 30 min, and the optical density of the resulting colored product in each well was measured at 405 nm with a microplate reader (BioRad, model 450). The absolute concentrations of IgG in the samples were determined relative to the standard curve. At autopsy, the presence or absence of tumors was recorded. Tumors were excised at autopsy and preserved in a solution of 10% formalin dissolved in PBS. Tissue samples from all groups were fixed and stained with hematoxylin and eosin at American Histolab, Inc. (Gaithersburg, MD). Pathological evaluation of slides was performed by Pathco, Inc. (Ijamstown, MD). Most tumors were diagnosed as squamous cell carcinoma. In a few instances the squamous cell carcinomas were accompanied by primary mammary carcinomas. Tumor incidence was defined as the number of animals with palpable and histologically verified tumors per number of animals injected. DATA ANALYSES analysis of variance was used to test comparisons among groups for each experimental parameter. Individual pairwise comparisons were analyzed with independent twotailedt tests. Because these comparisons were planned, no post hoc corrections were used (Keppel, 1982). Some data were nonparametric, and mean differences were evaluated with x2. The treatment effects were considered statistically significant if p < 0.05. An overall EXPERIMENT 1 adult female deer mice were housed individually in either LD 16:8 or LD 8:16 for 8 weeks as described above. Half of the animals were then injected s.c. with DMBA or the DMSO vehicle, and were palpated weekly for tumors. Eight weeks after injection, the animals were treated as described above. Sixty-four EXPERIMENT 2 days decrease plasma estradiol levels. If DMBA tumors are estrogen-dependent, then exposure to short days may reduce tumor development by suppressing estradiol levels. To determine whether the short-day effects on tumorigenesis and immune function are mediated by estradiol, we maintained 120 adult female deer mice in LD 16:8. Animals were assigned to one of 10 experimental groups (n 12/group). Females were subjected to either a bilateral 50 days of age. Ovariectomized animals either (1) ovariectomy or a sham operation at received no further treatment, (2) received weekly injections of estradiol-17 P (Sigma; 0.11 mg) dissolved in 0.1cc of sesame seed oil, or (3) received weekly injections (0.1cc) of Short = the oil vehicle alone. Sham-ovariectomized females either (1) received no further treatment or (2) received weekly s.c. injections of sesame seed oil. One week after the injection regimen began, half of the animals in each of the five groups were injected s.c. in the lower left abdominal region with DMBA dissolved in DMSO (50 mg/kg), and half of the animals were injected with the DMSO vehicle alone. All animals were palpated on a weekly basis for tumors for 8 weeks, then killed and examined as described above. 237 Two additional groups of adult female deer mice were maintained individually in LD 16:8. Both groups of females were ovariectomized at 50 days of age; one group (n 12) received weekly s.c. injections of estradiol, and the other group (n 10) received injections of the sesame seed oil vehicle as described above. After 8 weeks, all mice were injected intraperitoneally (i.p.) with SRBC to assess antibody formation. A blood sample was obtained 4 days after SRBC injection, and IgG titers were assayed by the ELISA techniques described above. = = EXPERIMENT 3 In addition to reducing levels of sex steroid hormones, exposure to short daylengths also reduces blood prolactin levels in every mammalian species thus far examined (Goldman and Nelson, 1993). Prolactin has pronounced effects upon immune function in a variety of species (reviewed in Welsch, 1985; Bernton et al., 1988). The effects of DMBA are believed to be partially dependent on estrogen and, more specifically, on prolactin. An elevation in the blood level of prolactin potentially increases tumor incidence (Welsch and Nagasawa, 1977). If prolactin effectively stimulates the growth and incidence of tumors, then reduced prolactin levels should result in decreased tumorigenesis rates. If prolactin does not affect tumorigenesis, then suppression of prolactin should not affect tumor incidence. Furthermore, if suppressed prolactin levels are mediating the short-day enhancement of immune function in deer mice, then long-day animals in which prolactin is experimentally suppressed should have elevated antibody production upon the presentation of a specific antigen, as compared to long-day animals experiencing physiological levels of circulating prolactin. If suppressed prolactin levels are not causing the shortday enhancement of immune function, then all mice in this experiment should have comparable levels of immune function. Seventy-six adult female deer mice were individually housed for 8 weeks in long days (LD 16:8) in conditions similar to those described above. Because prolactin secretion is effectively suppressed by dopamine, a dopamine agonist, bromocriptine, was used to lower the blood prolactin concentrations (Neill and Nagy, 1994). Daily injection of bromocriptine was not a satisfactory method of prolactin suppression, because the repeated stress of the daily injections might affect immune function and potentially confound the results; weekly injections were considered preferable, in order to diminish stress responses. After 8 weeks in long days, half of the mice were injected s.c. with a suspension of microspheres providing a slow, regular release of bromocriptine (Sandoz, Basel, Switzerland; see Neidhart, 1989) in a dose of 125 p,g each day, for an initial period of 21 days (i.e., each suspension injected contained 125 f.Lg x 21 days 2.33 mg bromocriptine); the remaining animals in this study received a placebo suspension. The injections were repeated every 3 weeks. Three days after the initial injection with the microspheres, half of the animals in each group were injected s.c. in the lower left abdominal region with the chemical carcinogen DMBA dissolved in DMSO (50 mg/kg) or with the vehicle alone. Thus, four experimental groups were formed with 15 animals per group, receiving either (1) bromocriptine + DMBA, (2) bromocriptine + DMSO, (3) vehicle suspension + DMBA, and (4) vehicle suspension + DMSO. During an 8-week postinjection period, animals were palpated to detect tumor growth on a weekly basis. After 8 weeks a blood sample was obtained and assayed for prolactin by radioimmunoassay (RIA); however, the prolactin RIA data were inadvertently = 238 destroyed. Tumors, spleens, and uteri were dissected performed to verify the nature of the tumors. at autopsy, and histopathology was EXPERIMENT 4 Melatonin also influences tumor growth. Experimental and clinical reports indicate that there is a link between cancer development and pineal function (Bartsch and Bartsch, 1981; Lapin and Ebels, 1981; Blask, 1984; Hill and Blask, 1988). The data, however, are often inconsistent. In some cases, pinealectomy enhances tumor development; in other cases, pinealectomy inhibits tumor growth (for a review, see Blask et al., 1992). The conflicting data suggesting both anti- and procarcinogenic effects of melatonin and conflicting effects on immune function are reminiscent of the &dquo;antigonadal&dquo; and &dquo;progonadal&dquo; effects of melatonin on reproduction (Reiter, 1983). Experiment 4 investigated the role of melatonin in mediating the photoperiodic influences on immune function. If increased levels of melatonin in short photoperiodic conditions decrease the susceptibility to tumorigenesis, then females maintained in long photoperiodic conditions and treated with melatonin implants should also display a decline in the susceptibility to develop tumors. In contrast, in the absence of melatonin treatment, long-day mice should show an increase in tumor susceptibility. Thirty-four female deer mice were housed in long days (LD 16:8) at 21° ± 2°C, with uncontrolled humidity (50-60%). Implants of melatonin capsules induce reproductive regression in deer mice comparable to that observed in animals maintained in short daylengths (Lynch and Epstein, 1976; Petterborg and Reiter, 1983; Carlson et al., 1989). At 50 days of age, females were implanted s.c. with silastic capsules (15 mm in length, inner diameter 0.147 cm, outer diameter 0.195 cm) filled with either melatonin (Sigma) or cholesterol. These capsules provided melatonin that approximated physiological levels of short-day animals necessary to induce reproductive regression in deer mice (Lynch and Epstein, 1976). Three days after receiving the melatonin or placebo implant, animals were injected s.c. with DMBA (50 mg/kg) or with the DMSO vehicle. Animals were checked at weekly intervals for tumor development, and autopsies were performed 8 weeks after DMBA or DMSO injection as described before. = = RESULTS EXPERIMENT 1 of the animals that developed tumors were diagnosed with squamous cell but some mice also developed primary mammary carcinoma. None of the mice carcinoma, maintained in short days developed tumors 10 weeks after treatment with DMBA. In contrast, 89% (8/9) of the LD 16:8 animals treated with DMBA developed tumors within 3-4 weeks. None of the mice in either daylength developed tumors after DMSO treatment (Fig. 1). Both DMBA and DMSO irritated the skin, regardless of the photoperiod in which the animals were maintained. However, short-day females exhibited fewer skin lesions (60% vs. 80%; p < 0.05), as well as accelerated healing of the lesions, compared to long-day females (Fig. 2). Animals injected with DMBA displayed more severe wounds and a slower The majority 239 FIGURE 1. Percentage of female deer mice housed in long (LD 16:8) or short (LD 8:16) days that developed histologically verified tumors after injection with the chemical carcinogen DMBA dissolved in DMSO, or injection with DMSO alone. Number of animals in each treatment group is indicated in parentheses. pace of healing than animals injected with DMSO (Fig. 3). IgG titers did not differ significantly between photoperiodic treatment groups (LD 16:8, 0.0065 ± 0.002 fLg/ml; LD 8:16, 0.0119 ± 0. 003; p >0.05). These results indicate that photoperiod affects the susceptibility to chemically induced tumorigenesis. However, the nature of the mechanism underlying the photoperiodic effect on tumorigenesis remains unspecified. One possible explanation may be that the tumors caused by DMBA are prolactin- and/or estrogen-dependent (Tamarkin et al., 1981). Short days could inhibit tumors by suppressing these hormones. Alternatively, melatonin might either be directly or indirectly involved in the photoperiodic modification of tumorigenesis. Regardless of the mechanism, the results of this study suggest a potentially important functional role of photoperiod in disease onset and in healing processes. EXPERIMENT 2 The rate of tumorigenesis did not significantly differ as a result of estrogen treatment among the groups that were injected with DMBA (p > 0.05 in all cases) (Fig. 4). These results suggest that the presence or absence of estrogen does not differentially affect susceptibility to DMBA-induced tumorigenesis. The groups that had been exposed to DMBA displayed tumor development in 62.5% to 75% of the cases. None of the animals injected with DMSO developed tumors (Fig. 4). Surgical incisions required an average of 14 days to heal completely in long-day females (p < 0.05). The presence of ovaries did not affect this rate of healing (p > 0.05). The changes in uterine mass were consistent with the fact that the absence of estrogen results in a substantial decrease in uterine mass. All ovariectomized females displayed a 240 FIGURE 2. Percentage of female deer mice that developed lesions after injection with DMBA (hatched or DMSO (filled bars) in short (top panel) or long (bottom panel) days. Other symbols and conventions as in Figure 1. bars) marked decline in uterine mass (p < 0.00 in all cases). In contrast, ovariectomized females in which estrogen has been replaced through weekly injections maintained uterine masses that were statistically equivalent to those of ovary-intact females (p > 0.05) (Table 1). Splenic mass was not significantly affected in ovariectomized deer mice by the presence or absence of estrogen (p > 0.05). However, the presence of intact ovaries significantly affected splenic mass (p < 0.05); intact females had significantly smaller spleens (absolute and adjusted for body mass) than ovariectomized females (Table 1). Injection with DMBA did not affect spleen mass in ovariectomized females that received weekly injections with 241 FIGURE 3. Subjective ratings of the lesions of female deer mice that developed after injection with DMBA (hatched bars) or DMSO (filled bars): (0) no lesions, (1) barely detectable lesions, (2) mild lesions (<3 mm), (3) moderate lesions (3-5 mm), or (4) severe lesions (>5 mm). oil or no injection, or in females that received in each case; data not shown). sesame weekly estradiol injections (p > 0.05 EXPERIMENT 3 Injection with a suspension of bromocriptine resulted in tumor development among 24% of DMBA-treated females, whereas females injected with the vehicle suspension alone developed significantly more tumors after exposure to a single dose of DMBA (55.6%; p < 0.05). 242 Percentage of ovariectomized (OvX), estradiol-treated ovariectomized (OvX + E), and gonadally intact (S - OvX) deer mice housed in long (LD 16:8) days that developed histologically verified tumors in response to DMBA or DMSO treatment. Other symbols and conventions as in Figure 1. FIGURE 4. Again, none of the mice treated with DMSO developed tumors (Fig. 5). Treatment with bromocriptine did not affect uterine or splenic mass in any of the four experimental groups (Table 2). Bromocriptine treatment also failed to affect wound healing (p > 0.05). EXPERIMENT 4 Mice treated with melatonin did not develop tumors 8 weeks after injection with DMBA. In contrast, 66% of the placebo group displayed tumors within 3 to 4 weeks after DMBA treatment (p < 0.05). None of the mice in either group developed tumors after treatment TABLE 1. Mean (± SEM) Body Mass and Absolute and Relative of Ovariectomized and Sham-Operated Females Note. Data of DMBA- and DMSO-injected animals did not differ An asterisk (*) indicates statistically significant results. Splenic significantly and and Uterine Mass were combined for this table. 243 FIGURE 5. Percentage of bromocriptine- or placebo-treated female deer mice housed in LD 16:8 that developed histologically verified tumors in response to DMBA or DMSO injections. Other symbols and conventions as in Figure 1. with DMSO (Fig. 6). Splenic mass was not affected by DMBA or melatonin treatment (p 0.05) (Table 3). Uterine mass only differed among the melatonin-implanted animals. Females treated with melatonin and exposed to DMBA had significantly higher uterine masses compared to melatonin-treated females injected with DMSO (p < 0.005) (Table 3). Overall, DMBA treatment resulted in a significant elevation in uterine mass (p < 0.005) (Table 3). Wound healing rate was unaffected by melatonin (p > 0.05). > DISCUSSION Animals maintained in short daylengths did not develop tumors after treatment with the chemical carcinogen DMBA when given this agent at a dose that produced tumors in 89% of long-day cohorts. Exposure to short daylengths inhibits development of DMBA-induced tumors. Short days also may enhance immune function. Healing was more rapid in shortday mice as compared to long-day deer mice, suggesting a photoperiodic effect on cytokine activities; however, a direct test of immune function, IgG responses to SRBC, was not affected by daylength in the present study. Tumor development was unaffected by manipulations of blood levels of estrogen. Suppression of blood prolactin levels and chronic elevation of blood melatonin levels led to a significant reduction in tumor incidence among long-day deer mice. Manipulation of blood levels of estrogen, prolactin, or melatonin did not affect healing rates among long-day animals. These results are consistent with previous findings that melatonin generally enhances immune function and suppresses tumorigenesis (Maestroni and Conti, 1991; Blask et al., 1992). The extent to which chemically induced tumorigenesis and immune function are related or independent phenomena, and the extent to which compromised immune function may affect DMBA-induced tumors, remain unspecified. The results of the present study suggest that seasonal changes in healing rate could be mediated by photoperiodic regulation of estrogen levels in female deer mice. Further studies are required to assess this hypothesis, as well as to assess the effects of androgens on healing 244 TABLE 2. Mean (±SE3o Body Mass and Absolute and Relative Splenic and Uterine Mass of Intact Females That Were Injected with a Slurry of Empty Microspheres or Microspheres Filled with Bromocriptine processes. A seasonal change in healing responses might reflect adaptive increases in immune function (Blom et al., 1994). Presumably, differential healing rates reflect differential cytokine responses. We attempted to assay interleukin-2 (IL-2) and IL-6 levels in the blood of these deer mice, using two different commercial kits (one kit was monoclonal for laboratory strains of house mice, Mus; the other kit used polyclonal antibodies). However, both assays failed to detect deer mouse IL levels. Although most of the immune assays are based on Mus, this species is not an ideal animal model with which to pursue this research, because Mus do not show reliable responses to daylength or melatonin. Further studies on the mechanisms of photoperiodic changes in healing processes await development of reliable cytokine assays in photoperiod-responsive species. Seasonal changes in the rate of DMBA-induced tumorigenesis in deer mice are estrogenindependent, but may be mediated by melatonin directly or by melatonin acting upon other FIGURE 6. Percentage of melatonin- or placebo-treated female deer mice housed in LD 16:8 that verified tumors in response to DMBA or DMSO injections. Other symbols in Figure 1. developed histologically and conventions as 245 TABLE 3. Mean (±SE3o Body Mass and Absolute and Relative Splenic and Uterine Mass of Intact Females Implanted with a Silastic Capsule Filled with Either Melatonin or Cholesterol Note. An asterisk (*) indicates statistically significant results. physiological parameters such as prolactin secretion. Thus, melatonin may play a critical role in the mechanism through which photoperiod affects the risk of tumor development. This could have clinical significance. For example, several groups have reported a seasonal variation in the month of initial detection of human breast cancer, with maximum detection reported in spring and early summer (Lee, 1967; Cohen et al., 1983; Hartveit et al., 1983; Kirkham et al., 1985; Mason et al., 1985; Chlehoun and Gray, 1987). The pattern of tumor detection is most apparent in premenopausal women with estrogen-receptor-positive tumors (Mason et al., 1985). Women secrete quantitatively more melatonin in winter than in summer (Kauppila et al., 1987), and a hormonally mediated increase in tumor rate during the spring might be mediated by the cyclic variation in the pattern of melatonin secretion (Mason et al., 1985). The exact nature of the mechanism of melatonin’s oncostatic properties, however, remains unspecified (Blask et al., 1992). One way melatonin might affect tumorigenesis is through direct interaction with the target tissue by modifying the process of tumor initiation (Tamarkin et al., 1981; Blask et al., 1992). Alternatively, indirect effects of melatonin on the secretion of steroid hormones, or other hormones such as prolactin that are suppressed in response to short days (or the pattern of melatonin secretion), might be involved. Among women, diminished melatonin levels secreted during the long days of spring and summer might allow an increase of ovarian steroidogenesis (Kauppila et al., 1987) or the production of prolactin (Wright et al., 1986). Consequently, long-day melatonin release profiles may mediate an increase in tumor growth via higher circulating levels of estradiol or prolactin. This would be particularly likely to occur in hormone-responsive tumors (Mason et al., 1985). In the present study, low prolactin levels reduced tumor incidence from 56% to 24%. Assessment of blood prolactin levels will be required to evaluate whether prolactin levels are equally suppressed in bromocriptinetreated animals as in melatonin-treated females. In the current study, our prolactin RIA data were inadvertently destroyed. There was no significant effect of photoperiod or hormone manipulation on IgG titers in the present study. Although IgG levels begin to increase 4 days after SRBC exposure in laboratory mice, maximal IgG responses are not observed until 8-10 days after antigen exposure. Thus, photoperiod could exert an effect on antibody production that was undetected in the present study. The results of the present study indicate that no photoperiodic differences 246 in IgG titers occurred within 4 days after exposure to SRBC. Improved IgG and IgM ELISA techniques for deer mice are currently being developed in our laboratory. Taken together, our results indicate that the ontogeny of a chemically induced tumor can be affected by photoperiodic manipulation. Melatonin, either directly or through its actions on other physiological processes such as prolactin secretion, may mediate photoperiodic effects on DMBA-induced tumorigenesis. Healing rates of deer mice maintained in short daylengths were accelerated as compared to those for long-day deer mice, suggesting photoperiodic mediation of cytokine activities. The enhancement of healing rates and suppression of chemically induced tumorigenesis associated with exposure to simulated winter daylengths imply an adaptive immune response to short days that would proactively counteract winter stress-induced immunosuppression in nature. Further studies are required to assess other functional changes in response to photoperiod in both immune processes and tumor development, using other tumor models. Also, the clinical significance of photoperiodic mediation of immune function and tumor incidence in humans requires further study. ACKNOWLEDGMENTS We are grateful for the excellent technical support and advice of L. Tamarkin, J. Shiber, C. Moffatt, M. Labbe, J. Fine, and A. Wohn; we thank R. Bungiro for superior animal care. We are also grateful for a generous contribution of bromocryptine microsphere suspensions from Sandoz, Inc., Basel, Switzerland, and thank C. Moffatt for developing the IgG ELISA for deer mice. 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