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Regular article |
a Center for Perinatal Biology,
b Departments of Physiology and
c Pathology,
d Loma Linda University School of Medicine and Immunology Center, Loma Linda University Medical Center, Loma Linda, California 92350
e MCP Industries, Inc., Corona, California 91718
ABSTRACT
The present study tested the hypothesis that acute treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs fertility, disrupts the nocturnal melatonin rhythm, and suppresses lymphocyte function. Adult Siberian hamsters administered 2 or 100 µg TCDD/kg body weight/0.2 ml sesame oil had a delayed latency to first litter and an increased adult mortality compared to hamsters given 0.1 µg/kg or vehicle. Within 75 days of TCDD treatment, full reproductive capabilities were achieved. Moreover, the nocturnal melatonin rhythm was not disrupted in adults administered TCDD or in their progeny. Lymphocyte activity varied with respect to time of day and treatment. Lymphocyte proliferation was enhanced at night irrespective of TCDD treatment; during the day, 2 wk after the 2-µg/kg treatment, blastogenesis was reduced compared to that in the 0.1-µg/kg group or in vehicle-treated controls. In contrast, TCDD did not affect the mixed lymphocyte reaction in response to allogeneic antigen when assessed at 2 and 20 wk post-treatment. Thus, findings indicate that TCDD produced acute effects on fertility, mortality, and systemic lymphocyte proliferation, but long-lasting effects on specific aspects of reproductive, neuroendocrine, and immune cell functions were not observed.
circadian rhythm, melatonin, pregnancy, reproductive immunology
INTRODUCTION
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is recognized to be teratogenic, embryotoxic, and carcinogenic [1]. The acute lethality dose for TCDD is among the lowest reported for a chemical substance. TCDD acts as an endocrine disrupter to alter differentiation and function of the reproductive system [2, 3]. Reproductive effects associated with high levels of TCDD during gestation are not tissue specific but may relate to effects on embryonic epithelial cells of ectodermal origin [3] and induce early fetal loss in primates [4, 5]. The immune system is one of the most sensitive targets for TCDD toxicity. As reviewed by Holsapple et al. [6], TCDD exposure inhibits aspects of innate immunity, such as complement activation, and adaptive immunity including cell-mediated immunity, T- and B-cell differentiation, and lymphocyte maturation, as well as increased susceptibility to infectious agents or tumor promotion. Natural killer cell and macrophage activities appear to be spared. Selectivity of TCDD effects on T-lymphocyte function includes suppression of the mixed lymphocyte reaction [7]. Studies of TCDD exposure in laboratory animals and in humans following accidental exposure [8–10] have yet to extend to seasonal breeders even though environmental contamination has a profound impact on life-cycle functions in animals in the wild [11].
For most mammalian species, a variety of physiological adaptations anticipate alterations in climate and nutrition and parallel changes in environmental photoperiod. The mechanism mediating the effects of photoperiod on reproduction and metabolism is known to involve an endogenous biological clock and the pineal gland hormone melatonin [12]. In contrast to inbred animal models where little is known about the role that melatonin plays in regulating physiology, the nocturnal melatonin rhythm is a sentinel cue in the neuroendocrine mechanism that controls seasonal reproduction and other physiological adaptations to changes in day length [12, 13]. Recent evidence in the Siberian hamster also suggests that photoperiod modulates a variety of immune cell functions, effects that may be mediated by the pineal gland [14, 15]. In other rodents, administration of melatonin stimulates important immune cell functions and suppresses tumor growth [16]. Moreover, TCDD is reported to suppress nighttime concentrations of melatonin in serum and its metabolite in urine [17, 18]. In the rat, circadian rhythms in prolactin, corticosterone, and thyroxine are suppressed or phase-delayed by a single 50-µg TCDD/kg body weight treatment [19]. These findings raise the possibility that TCDD may affect the biological clock mechanism that regulates circadian rhythms and, in turn, influence immune cell function. Whether TCDD affects the nocturnal rhythm of melatonin in the pineal gland or circulation, a commonly used marker for clock function, has not been studied in any species, nor have the effects of TCDD on fertility or select immune functions been addressed in a seasonal breeder. Thus, the present study in the Siberian hamster tested the hypothesis that acute exposure to TCDD alters fertility, the pineal melatonin rhythm, and select immune cell functions.
MATERIALS AND METHODS
General Procedures
Siberian hamsters (Phodopus sungorus) were maintained in long days from birth (16 h of full spectrum overhead fluorescent light, ~300 lux from 1900 to 1100 h PST) and used in this study as adults (>90 days of age). Temperature and humidity were constant at 25°C and 70%, respectively; food and water were continuously available. The Institutional Animal Research Committee approved all treatments and animal husbandry procedures. Body weights were measured for several weeks after TCDD treatment. When needed, about 0.5 ml of whole blood was obtained from the ocular sinus of each hamster with a heparinized microcapillary tube. Blood was drained into a 12 x 75-mm test tube for serum assays or, for immune cell tests, a 1.5-ml microcentrifuge tube that contained 0.1 ml of heparin (1000 U/ml). Mortality following this ocular bleed protocol is typically low; 4 of 115 hamsters (3.5%) were found dead within 24 h of ocular blood collection in this study.
Dioxin Treatments
TCDD (99% purity) was purchased from Cambridge Isotope Laboratories (Andover, MA). Adult hamsters were anesthetized with sodium pentobarbital (15 mg /kg body weight, i.p.), then administered a single dose of TCDD (either 0.1, 2, or 100 µg/kg, as specified below) in 0.2 ml sesame oil by gavage. Limited animal numbers precluded treatment of males at the 100-µg/kg TCDD dose. Vehicle-treated hamsters served as controls. Hamsters recovered from anesthesia under a warming lamp. Doses were selected based upon prior reports in rodents in which effects included an influence on sexual differentiation, reproductive development, reduced adult sperm counts, and effects on immune cell functions [6, 20]. Stringent safety procedures adhered to regulations by the California Environmental Protection Agency to prevent hazardous exposure to laboratory and ancillary personnel.
Assessment of Reproductive and Neuroendocrine Function
Breeding study Mating pairs of male and female hamsters were set up 10 days after treatment with TCDD (n = 34) or vehicle (n = 16). In the 0.1-µg/kg group, TCDD-treated hamsters were paired with a conspecific that was treated with vehicle (n = 7, 4 male and 3 female pairs) or the same dose of TCDD (n = 3). In the 2-µg/kg group, TCDD-treated hamsters were paired with a conspecific that was treated with vehicle (n = 8, 5 male and 3 female pairs) or the same dose of TCDD (n = 4). Within each group, data were not statistically different with respect to sex (P > 0.05, t-test). In the 100-µg/kg group, 10 females were administered TCDD then, 10 days later, paired with vehicle-treated males. The colony was observed each day during the morning and afternoon to assess morbidity, mortality, latency between initial pairing and day of birth, litter size, and pup viability. Progeny were weaned at 3 wk of age, earmarked, and recruited as adults to test the hypothesis that TCDD treatment of parents impairs the nocturnal melatonin rhythm.
Melatonin rhythms An ocular blood sample was obtained from each hamster 7 days following treatment with TCDD or vehicle, i.e., at specific times before or after lights-off, to assess the nocturnal melatonin rhythm (n = 4–7/group/time). When progeny of TCDD-treated animals were adults (3 mo of age), this same blood sampling protocol was used to assess the nighttime melatonin rhythm. Melatonin concentrations were determined by RIA as previously described [21]. Assay sensitivity was 16 pg/ml (two standard deviations from buffer controls). Inter- and intra-assay coefficients of variation averaged <15% (n = 6 assays).
Assessment of Immune Cell Function
Spontaneous lymphoblastogenesis To assess basal lymphocyte proliferation, ocular blood samples were obtained 7 days after TCDD or vehicle treatment, at 2 h before or 4 h after lights-off (n = 5–6/group). A 0.05-ml aliquot of blood was assayed in triplicate as previously described [14, 22].
Mixed lymphocyte reaction This test was performed to assess the capabilities of lymphocytes from control and TCDD-treated hamsters to recognize alloantigen. Lymphocytes were isolated from a 0.5-ml ocular blood sample by Ficoll-Hypaque density gradient [23]. Use of an animal Ficoll preparation (1-Step 1.077/265, Accurate Chemical and Scientific Corp., Westbury, NY) improved lymphocyte yield over the human Ficoll preparation (Lymphocyte Separation Solution; The American Red Cross, Washington, DC). Lymphocytes from TCDD- and vehicle-treated hamsters (1 x 106 cells) were incubated with allogeneic stimulator cells (3 x 106 cells), i.e., an aliquot of pooled irradiated splenic lymphocytes (3000 rad for 6.5 min) from four untreated Siberian hamsters. This radiation dose was empirically determined to inhibit proliferation by hamster lymphocytes. These cells were cocultured (RPMI-1640 with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin) for 5 days and then pulsed for 18 h with [3H]thymidine (1 µCi/0.02 ml media). Cells were harvested on glass filter mats and uptake of radioactivity assessed by beta scintillation.
Statistical analysis In the breeding study, mating groups were defined by the highest dose of TCDD administered to a hamster in the pairing because data were not statistically different with respect to sex or TCDD dose (P > 0.05, t-test). Serum and pineal melatonin data were log-transformed to normalize variance, then evaluated by two-way analysis of variance (time versus treatment; SPSS, Chicago, IL) followed by Duncans multiple range test for individual comparisons. When Levenes test for homogeneity of variance was significant, nonparametric tests including the Kruskal-Wallis and Mann-Whitney U-test were used. Other data were evaluated by analysis of variance or t-test. P < 0.05 was considered significant. Results are presented as mean ± SEM.
RESULTS
Mortality
In daily observations, acute administration of TCDD did not obviously diminish general health or activity of hamsters over the 20-wk study. Body weights were unchanged in individuals and among groups during the weeks after TCDD treatment (data not shown). Over the 20-wk study, deaths occurred intermittently (Fig. 1). A greater than twofold increase in mortality occurred in hamsters treated with the 2- and 100-µg/kg doses of TCDD compared to vehicle or 0.1-µg/kg groups by the conclusion of the study. Of the 105 TCDD-treated hamsters, 22 died. Deaths in the 100-µg/kg group were associated with a bloody vaginal discharge and resorbing fetuses of a failed pregnancy (n = 2) and a fighting injury (n = 1). All other hamsters were found dead; the cause, for the most part, was not known. The 8% mortality in vehicle-treated controls was not unusual (n = 6) given group housing conditions and novel mating. Even among those that died, nonspecific dermatological symptoms, i.e., chloracne, skin discoloration, or hair loss were not evident in hamsters. Data from hamsters that died during the course of the study were excluded from further analyses.
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Reproductive and Neuroendocrine Functions
Breeding study Acute treatment with TCDD reduced fertility but effects were dose and time dependent. Elapsed time between pairings in the 0.1-µg/kg group and birth of the first litter averaged 24 days (Fig. 2), i.e., 34 days after TCDD administration. This latency to first litter was not statistically different from that in controls. By contrast, the mean latency to produce a litter was significantly delayed, to 49 days after mating, in hamsters treated with either the 2- or 100-µg/kg dose. These doses of TCDD did not permanently block fertility (Fig. 3). Litters produced by hamsters given the 2- or 100-µg/kg dose occurred gradually over the subsequent 30-day period. On average and typical of controls, four pups were born per litter (approximately equal sex ratio); all were weaned and survived into adulthood irrespective of parental TCDD treatment. Eventually, pups were born to all pairs by 75 days after mating compared to the prevalence of births within 20 days of mating in controls and by 22 days in the 0.1-µg/kg group.
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Melatonin rhythms One week after treatment, nocturnal melatonin rhythms were evident in all groups (Fig. 4). Serum melatonin concentration were significantly increased within 3 h after lights-off and remained elevated for the duration of darkness in TCDD-treated hamsters and vehicle controls. At some times, circulating melatonin concentrations were increased in hamsters treated with TCDD compared to that in controls (P < 0.05). However, neither the duration nor peak amplitude of the nighttime rise differed consistently with respect to treatment. Melatonin rhythms were also determined in progeny of TCDD-treated hamsters at 3 mo of age (data not shown). No statistically significant differences in serum melatonin concentrations were evident at any time in adult progeny from parents that had been given TCDD.
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Immune Cell Functions
Spontaneous lymphoblastogenesis Lymphocyte proliferation in vitro varied with respect to in vivo TCDD treatment and time of day when blood was obtained (Fig. 5). One week after treatment, proliferation of lymphocytes from hamsters given 2 µg/kg TCDD was suppressed more than fivefold during the day compared to that from hamsters given vehicle or the 0.1-µg/kg dose. At night, lymphoblastogenesis was over fourfold greater than that during the day in all groups; no statistical differences were found between groups of hamsters administered vehicle or TCDD treatment.
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Mixed lymphocyte reaction Stimulation of lymphocyte proliferation in response to alloantigen was not significantly different in hamsters given TCDD compared to that in vehicle-treated controls (Fig. 6). As a percentage relative to controls, the in vitro proliferation responses were equivalent in hamsters at 2 and 20 wk following in vivo TCDD or vehicle treatment.
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DISCUSSION
Studies were designed to test the hypothesis that an acute single exposure to TCDD alters reproductive capability, the pineal melatonin rhythm, or select immune cell functions in the Siberian hamster. Evidence indicated that TCDD treatment forestalled reproduction, but effects were dependent upon dose administered and were transitory. Females in the 2- and 100-µg/kg dose groups failed to conceive within 3 wk after pairing, a time when vehicle-treated and the 0.1-µg/kg groups typically produced litters. A period of infertility lasted for nearly 6 wk in hamsters given the highest TCDD doses; gestation is 19 days in this species. No long-term effects of TCDD treatment on litter size or pup viability were observed during the 20-wk study. TCDD also did not disrupt the nocturnal melatonin rhythm in treated hamsters or their adult progeny. Assessment of immune cell activities indicated no effect of TCDD treatment on spontaneous lymphocyte proliferation at night or the capabilities of lymphocytes to recognize allogeneic antigen in culture. Overall, the data suggest important short-term consequences of a single TCDD treatment in the hamster but no long-term repercussions on reproduction, neuroendocrine, or immune system functions.
Acute consequences of TCDD treatment in the present study may be related to dosage. TCDD treatments as low as 2 µg/kg suppressed fertility or immune cell function, an effective dose that was comparable to that used previously to impact reproductive function and development [3]. By comparison, the 0.1-µg/kg dose of TCDD consistently had little or no effect on experimental parameters in the study. This lower dose is roughly equivalent to the medium exposure following a thermochemical accident [24] and is some 100-fold higher than that reported for adults in industrialized countries. Background levels have been estimated to be 26 pg/day, primarily through daily consumption of animal-derived food sources [7] and 7–12 ng/kg body weight [25]. Such ambient environmental contamination produced 6 ppt TCDD/ml serum in humans and was associated with few health risks compared to the profound increase in specific diseases or immunological changes among workers following accidental exposure to TCDD [8–10].
Fertility, Mortality, and Neuroendocrine Effects of Acute TCDD Treatment
A major consequence of TCDD treatment was a profound though transitory influence on reproductive capabilities. The 2- and 100- but not 0.1-µg/kg treatment delayed fertility. Increased latency to the first litter raises the possibility that the TCDD could directly and acutely affect reproductive organs or central sites that are involved in the neuroendocrine control of ovulation [26]. Irregular cycles, reduced ovulation rate, and increased pre- and postimplantation loss occur in female rats given 2 µg/kg [27]; higher doses had more pronounced effects. Whether impaired mating or fetotoxicity contributed to reduced fertility following TCDD treatment in the present study cannot be excluded from consideration. Thus, further work is needed to resolve the specific site of action for TCDD among various components of the reproductive system.
In addition, restricting the focus for TCDD action to the female does not explain why fertility was delayed to the same extent when only males were given the 2-µg/kg dose; again, the 0.1-µg/kg dose was without effect. In other rodents, a similar TCDD dose range during development significantly reduced ejaculated sperm counts and epididymal sperm storage by 38% in adulthood [3, 19, 28]. Thus, TCDD treatment may temporarily decrease sperm counts and acutely impair spermatogenesis. However, the transitory effects of TCDD on fertility may also involve a mechanism common to both partners in the breeding pair. Chahoud et al. [29] suggest that TCDD may affect the process of fertilization even when males were treated with TCDD prior to mating. Moreover, a dose of 2 µg/kg increases pre- and postimplantation loss in the rat [27]. Regardless of the locus of action, lack of a long-term consequence of acute TCDD treatment may reflect a diminishing tissue-specific body burden, an effect that is likely to decrease over time based upon the estimated half-life for TCDD of approximately 4 wk [1].
In contrast to major effects of acute TCDD exposure on fertility and mortality, the present findings do not support the hypothesis that acute TCDD exposure influenced the timekeeping mechanism that generates the nocturnal melatonin rhythms. By comparison in the rat, nocturnal serum melatonin is reduced more than 50% after acute treatment with TCDD [17, 18]; duration of the nighttime rise was not assessed. Moreover, TCDD exposure was associated with reduced nighttime urinary excretion of the melatonin metabolite 6-hydroxy melatonin sulfate and a lack of change in increased pineal melatonin production at night. Why nighttime melatonin concentrations in circulation and its metabolism are both reduced when pineal production remains unchanged following TCDD treatment was not clear. However, other evidence suggested that extrahepatic metabolism of melatonin was enhanced by the acute TCDD treatment. Thus, these finding are consistent with those in the present report and do not suggest that TCDD impairs clock-mediated drive of nighttime melatonin production.
The biological relevance of reported effects of TCDD treatment on the melatonin rhythm is further questioned because the importance of amplitude of the nighttime melatonin rise for any physiological function has yet to be established for any species [13]. Studies in which melatonin concentrations were assessed at only a single time of night cannot resolve whether a day/night difference in circulating melatonin persisted after TCDD treatment or account for inherent variability in the nocturnal melatonin rhythm. Occasional statistical differences in melatonin concentrations between TCDD- and vehicle-treated hamsters in the present study may reflect normal variability in this circadian rhythm as is evident even in replicate experiments or with repeated samples from the same individual [30, 31]. Rather, duration is considered the crucial feature of the melatonin rhythm that reflects endogenous clock function and serves as a physiological representation for the period of darkness [12, 13]. In the present study, duration of the increase in pineal and serum melatonin at night was comparable in TCDD-treated hamsters and vehicle controls or their progeny. Thus, the data support the conclusion that acute TCDD exposure does not interfere with the complex neuronal mechanisms that contribute to biological timekeeping. These results do not exclude the possibility that TCDD may affect entrainment, phase response to stimuli, or other fundamental timekeeping mechanisms that are related to adaptations to a relevant photoperiod challenge.
Immune Effects of Acute TCDD Treatment
The effect of TCDD on spontaneous lymphocyte proliferation was dependent on dose and time of day when blood was collected. Acute exposure to TCDD in vivo, at the 2-µg/kg but not the 0.1-µg/kg dose, suppressed in vitro lymphoblastogenesis during the day but not night. This immunosuppressive effect of TCDD occurred when basal proliferation was already low and did not extend to the night when hamsters are typically in the active phase of the circadian cycle. Whether reduced lymphoblastogenesis during the day, 1 wk after acute TCDD treatment, may be due to low serum melatonin concentrations or another component of the endocrine milieu is not known. Although exogenous melatonin enhances lymphocyte proliferation in other rodents [16], the present findings also do not directly address the possibility that increased concentrations of melatonin in circulation at night counteract the effect of TCDD to reduce basal lymphoblastogenesis during the day. However, this possibility is not supported by the finding that melatonin, albeit at a pharmacologic dose, suppressed proliferation by human lymphoid cells [32].
Another test of lymphocyte response capabilities was used to assess the proliferative capabilities of T-helper and precursor T-cytotoxic cells to respond to allogeneic lymphocytes [22, 33, 34]. Results indicate that the mixed lymphocyte stimulation index was similar among hamsters given vehicle or TCDD at both 2 and 20 wk after treatment. Thus, lack of a treatment-dependent or long-term difference in the mixed lymphocyte culture response further suggests that acute dosing with TCDD did not irreversibly impair the ability of lymphocytes to recognize and respond to alloantigens.
In summary, this is the first report to provide information on the effects of TCDD on reproduction, biological timekeeping, and immune cell function in a seasonal breeder. Such studies of circadian and immune cell functions are important to assess normative physiological adaptations that are regulated by photoperiod. Findings indicate that fertility was transiently impaired by acute exposure to TCDD at concentrations that produced effects in other rodent species. However, reproductive capabilities fully recovered within 7 wk of treatment. During this period, TCDD treatment failed to alter the nocturnal melatonin rhythm, a sentinel cue for the endogenous timekeeping and an effector signal of the neuroendocrine mechanism that controls reproduction in the hamster. Effects of TCDD on select cell-mediated immune functions varied with dose and, for lymphocyte proliferation, time of day when samples were collected. In one respect, absence of TCDD treatment effects on nighttime lymphoblastogenesis and mixed lymphocyte reactivity to alloantigen do not suggest extensive or prolonged immune system suppression. By contrast, effects of TCDD on daytime lymphocyte proliferation and fertility indicate that additional endpoints are needed to assess fully the consequences of TCDD exposure. Further insight may come from investigations of other doses and more extended TCDD exposures on reproductive, neuroendocrine, or immune function in studies that focus on photoperiod control of circadian immune cell functions.
ACKNOWLEDGMENTS
We thank Long Tran for technical assistance in the conduct of the experiment and preparation of graphs. Critique of this work at several stages of this project by Dr. Linda Birnbaum is deeply appreciated.
FOOTNOTES
First decision: 31 January 2000.
1 This effort was supported, in part, by MCP Industries, Inc., Corona, CA. 
2 Correspondence: FAX: 909 824 4029; syellon{at}som.llu.edu
Accepted: March 28, 2000.
Received: December 22, 1999.
REFERENCES
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