* Toxicology Group, Wageningen University, Wageningen, The Netherlands; Laboratory Animal Center, Wageningen University, Wageningen, The Netherlands;
Department of Animal Physiology, Wageningen University, Wageningen, The Netherlands;
Department of Environmental Chemistry, Wallenberg Laboratory, Stockholm University, Stockholm, Sweden; ¶ Institute of Environmental Studies, Vrije Universiteit, Amsterdam, The Netherlands
Received May 31, 2004; accepted August 6, 2004
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ABSTRACT |
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Key Words: estrous cycle; anogenital distance; PCB; metabolite; reproductive senescence.
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INTRODUCTION |
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Binding of OH-PCBs to TTR in vivo is thought to facilitate the transport of OH-PCBs across the placenta, thereby affecting maternal, but especially fetal, thyroid hormone levels (Brouwer et al., 1998). Earlier studies showed a selective accumulation of the PCB metabolite 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107) in fetal and neonatal rats after maternal exposure to Aroclor 1254 from gestation day (GD) 10 to GD 16 (Morse et al., 1996a
). Thyroid hormone levels in the exposed fetuses and neonates were significantly decreased, and it was hypothesized that this decrease was caused by the competitive binding of 4-OH-CB-107 to TTR, which was observed for this compound in vitro (Meerts et al., 2002
). We were able to support this hypothesis in an in vivo study, where exposure of pregnant rats to 14C-4-OH-CB107 from GD 10 to GD 16 resulted in accumulation of 4-OH-CB107 in fetal livers, brain, and plasma measured at GD 17 and GD 20. Polyacrylamide gel electrophoresis of maternal and fetal plasma revealed the binding of 4-OH-CB107 to TTR. Consequently, maternal, but especially fetal, total thyroxine (TT4) levels at GD 20 were significantly decreased by 38% and 89%, respectively (Meerts et al., 2002
).
A prenatal or early postnatal hypothyroid state is known to severely affect the normal development of the brain and sexual organs. Effects on brain development include disorders of neuronal process growth (Stein et al., 1991), disruption of the expression pattern of neurotrophins, nerve growth factor, and brain-derived neurotrophic factor (Nevue and Arenas, 1996
), as well as interference in neurotransmitter systems (Seegal, 1996
). Exposure of rats to Aroclor 1254 resulted in alterations in regional brain serotonin metabolism and in glial and neuronal cell markers (Morse et al., 1996b
). Exposure of rats to Aroclor 1016 from GD 8 through weaning caused elevations in regional dopamine concentrations in rat offspring. Studies with individual PCB congeners revealed that the structure of the congener and the age of the animal at the time of exposure were important variables for the observed effects on brain dopamine levels.
In addition to the above-mentioned effects of PCB-induced hypothyroidism on brain development, it is also possible that the relatively high concentrations of OH-PCB congeners in plasma or brain of fetal rats have a direct effect on brain development and/or reproduction. OH-PCBs are known to induce uncoupling of oxidative phosphorylation in mitochondria (Narasimhan et al., 1991), and inhibition of intercellular communication (de Haan et al., 1994
). Some OH-PCBs also possess (anti-) estrogenic activities (Korach et al., 1988
, Moore et al., 1997
). The OH-PCBs identified in human serum were mostly anti-estrogenic (Moore et al., 1997
). Kester et al. (2000)
reported extremely potent inhibition of human estrogen sulfotransferase activity in vitro by environmentally relevant OH-PCBs, suggesting that OH-PCBs indirectly induce estrogenic activity by increasing estradiol bioavailability in target tissues.
The aim of the present study was to investigate the potential impact of in utero exposure to 4-OH-CB107 on the development of rat offspring and the possible long-term effects on sex steroid hormone levels and reproduction. Effects on thyroid hormone concentrations and brain development were also investigated, but are reported elsewhere (Meerts et al., 2004). Pregnant rats were exposed to 0.5 or 5 mg 4-OH-CB107 per kg bw from GD 10 to GD 16. Exposure of pregnant rats to a concentration of 5 mg 4-OH-CB-107 per kg bw per day from GD 10 to GD 16 resulted in a concentration of 4-OH-CB107 in the fetal compartment comparable to the concentration that was reached after in utero exposure to Aroclor 1254 (Meerts et al., 2002
; Morse et al, 1996a
). In fetal plasma at GD 20, concentrations of 37.2 ± 5.14 nmol/ml were measured after in utero exposure to 4-OH-CB107 (Meerts et al., 2002
). A concentration of 0.5 mg/kg bw per day was additionally chosen to determine possible dose-dependent effects of 4-OH-CB-107. To determine if the effects observed in offspring exposed to Aroclor 1254 (as described by Morse et al., 1996a
) can be explained mainly by the accumulation of the metabolite 4-OH-CB107 in fetuses and neonates, an additional group of rats was exposed to Aroclor 1254.
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ANIMALS, MATERIALS, AND METHODS |
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Animals and treatment. All experimental procedures involving animals were approved by the Animal Welfare Committee of Wageningen University. Wistar WU rats (60 females, 30 males; 14 weeks old) were purchased from Charles River (Sulzfeld, Germany) and allowed to acclimatize for 3 weeks. Throughout the experiment, animals were kept in an artificial 12 h:12 h lightdark cycle with lights on at 06:00 h. Room temperature was maintained at 21° ± 2°C and humidity at 50 ± 10%. Animals were provided rat chow (Hope Farms, Woerden, the Netherlands) and tap water ad libitum.
After the acclimatization period two females were placed in a cage with one male from 17:00 to 8:00 h the next day. Females were examined for copulation each morning by checking the presence of sperm in the vaginal smear. When spermatozoa were found, that day was designated as day 0 of gestation (GD 0), and females were housed individually. On GD 10 the pregnant rats were divided randomly into the different treatment groups and transferred to a Macrolon, stainless steel cage to facilitate the collection of PCB-contaminated feces. In total 52 pregnant rats (13 per exposure group) were dosed by oral intubation with 0 (corn oil), 0.5, or 5 mg 4-OH-CB107 per kg body weight per day dissolved in corn oil (2 ml/kg) from GD 10 to GD 16. For comparison of the effects of 4-OH-CB107 with effects caused by parent PCB congeners, a fourth group of rats was dosed with 25 mg Aroclor 1254 per kg body weight from GD 10 to GD 16. In an earlier study it was observed that this dose level of Aroclor 1254 resulted in an amount of 4-OH-CB107 in the fetal compartment in the same range as observed after in utero exposure to 5 mg/kg 4-OH-CB107 (Meerts et al., 2002).
Maternal body weights were monitored daily throughout gestation. On GD 20, pregnant females were transferred to bedding material. At birth, i.e., postnatal day (PND) 0, live offspring were counted and sexed. Individual pups and dams were weighed on PNDs 1, 4, 7, 14, and 21, and after weaning body weights of the offspring were monitored weekly until sacrifice. On PND 4, litters were adjusted to 4 males and 4 females. Generally, this required the termination of excess offspring. However, in a few cases where a litter contained fewer than eight pups or the sex distribution was not permissive, the standardized litter required pups from two dams. To maintain litter independence, no dam was allowed to contribute pups to more than one litter. In addition, pups transferred from one litter to another were not used for any analysis. The standardized litter became the experimental unit and all treatment mean values reported are litter based. Liver, kidneys, brain, and thymus of the excess offspring were weighed. The remaining offspring were numerically marked on their feet to identify individual animals within a litter.
Developmental landmarks. During the study, a number of developmental landmarks for all litters were recorded in a blind fashion. Treatment groups were decoded only after termination of the animals when all analyses were completed. On PND 1 and PND 4, anogenital distance (AGD) and crownrump length (CRL) were measured on each pup by means of a micrometer capable of resolution to 0.01 mm. The AGD was measured in both sexes as the distance from the anterior edge of the anus to the base of the genital tubercle. Measurements of AGD and CRL were done by one person, to avoid interindividual variations. Each pup was additionally examined for the following developmental landmarks: pinna detachment (starting on PND 1), age at the onset of hair growth, and age at bilateral eye opening (starting on PND 12).
Following weaning at PND 21, pups were housed with littermates in unisexual groups, two pups per cage. Dams were sacrificed at PND 21 and blood was collected via the vena cava in heparinized tubes for thyroid hormone measurements. Liver, kidneys, adrenals, thymus, brain, spleen, uterus, and ovary were collected, blotted dry, and weighed. All organs were frozen in liquid nitrogen and stored at 80°C.
Female pups were examined daily for vaginal opening, starting at PND 30. The age at preputial separation (Korenbrot et al., 1977) in male offspring was examined from PND 35 until a complete preputial separation in all males was achieved. After puberty, the offspring were split into two cohorts; one cohort (n = 41 litters with 2 males and 2 females per litter) was housed individually and used for behavioral studies (Meerts et al., 2004
). From the other cohort (n = 41 litters, 2 males and 2 females per litter), females were also housed individually to study estrous cyclicity and reproduction as described below. Male offspring from this cohort were housed in unisexual groups with 2 animals per cage until dissections at about 11 months of age.
Reproductive capability of female offspring. Female vaginal estrous cyclicity was monitored for 21 days, starting at PND 210, by daily evaluation of the vaginal smears (between 8:00 and 10:00 h and at other times as needed). Differentiation of the cells during the 4 days of the estrous cycle was determined microscopically according to the method of Staples and Geils (1965). Because of the effects found on the length of the estrous cycle (cf. Results), a study was conducted to determine the reproductive capability of the female offspring. The females were split into two cohorts; one cohort stayed in unisexual groups with 2 females per group until necropsy at about 11 months of age to determine possible long-term adverse effects on sex steroid parameters. The other cohort (n = 41) was housed individually, and after 2 weeks mating with untreated males (16 weeks old, Charles River, Sulzfeld, Germany) was started (1:1). Females were examined for copulation each morning by checking the presence of sperm in the vaginal smear. When spermatozoa were found, that day was designated as GD 0. If no spermatozoa were found, the female was remated up to a maximum of 2 weeks with a stud male. The number of matings was recorded. Pregnant females were sacrificed at GD 20. Maternal blood was collected via the vena cava in heparinized tubes, and plasma was prepared and stored at 80°C for measuring thyroid hormones, estradiol, progesterone, and testosterone. Maternal body weight, ovarian weights, and the number of corpora lutea (examined with a microscope) implantation sites and embryos were recorded. Additionally, maternal liver, kidneys, spleen, brain, and thymus were weighed. From the fetuses, sex was determined and liver, brain, and thymus were removed and weighed.
Long-term effects on male and female hormone levels. Male offspring and the female cohort that was not used for the reproductive capability study were dissected at about 11 months of age (PND 310320) to determine possible long-term adverse effects on sex steroid parameters. To avoid the effects of stress on serum steroid hormone levels, the animals were killed by decapitation within 15 s of removal from their cages. Dissections were conducted between 08:00 and 12:00 to minimize circadian influences on testosterone and estradiol levels. Trunk blood was collected in Eppendorf tubes (for serum preparation) and heparinized tubes (for plasma) for hormone analysis. Immediately after collection of the blood, the brains were dissected rapidly (within 5 min) and separated in different regions for neurotransmitter analyses (Meerts et al., 2004). Weights measured at dissection included body, liver, kidneys, adrenals, spleen, thymus, and pituitary gland. From the males, testes, prostate, seminal vesicle, and cauda epididymis were weighed additionally. The coagulating glands were detached from the seminal vesicles and care was taken to avoid expression of fluids from these organs. From the females, uteri and ovaries were weighed. Estrous cycle stage was estimated at dissection from the appearance of the uterus as either estrous ("ballooning") or nonestrous.
Measurement of sex steroids. Before estradiol measurements, 100 µl plasma was extracted three times with 1.25 ml dichloromethane in glass tubes by vortexing for 30 s, centrifugation at 1,000 x g for 5 min, and collection of the dichloromethane phase. The dichloromethane phases were pooled, evaporated to dryness in a Savant Speed Vac vacuum concentrator, and 175 µl phosphate buffered saline (PBS) containing 0.1% (w/v) BSA was added to each tube. After thorough vortexing, estradiol concentrations were measured in triplicate as described by Palm et al. (1999). The extraction efficiency, determined by the addition of tracer amounts of 125I-E2 before extraction of the plasma, was 94.2 ± 2.84% for plasma from pregnant rats and 95.5 ± 2.32% for non-pregnant rats.
Progesterone concentrations were measured in triplicate in unextracted plasma, diluted 20 times in PBS containing 0.1% (w/v) BSA, as described by Van der Meulen et al. (1988) and Mattheij et al. (1995). Testosterone concentrations were measured in duplicate in extracted serum using a commercial ELISA kit (DRG, Marburg, Germany). Testosterone was extracted from 200 µl serum using the extraction method described above for estradiol analysis. The extraction efficiency was determined by comparing extracted standard samples with the non-extracted standards and was 95.3 ± 0.6%.
Statistical analysis. Statistical analysis was performed using the SPSS statistical software package. Differences between the number of pups and organ weights were analyzed by means of analysis of variance (ANOVA). Levene's test was used to evaluate homogeneity of variances, and the Bonferroni test was used to compare individual treatment means when ANOVA indicated that significant differences were present. For the evaluation of body weight development until sacrifice, ANOVA with repeated measures was used, with age as the factor. For hormone determinations, one pup per litter was used. Nonparametric analysis used the Kruskal-Wallis ANOVA by ranks. When this indicated significant differences, treatment ranks were compared to the control group by the Wilcoxon-Mann-Whitney test. Categorical data were analyzed by chi-squared analysis.
In all cases, the litter was the independent experimental unit, and data from individual male and female offspring were assumed to be representative for the litter. Where more than one male or female from a given litter was evaluated, the results were averaged to form a litter mean. In all cases significance was set at p < 0.05.
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RESULTS |
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The onset of bilateral eye opening was significantly earlier in male and female offspring exposed to Aroclor 1254 via the dams, as compared to the other exposure groups. The day of vaginal opening was not changed by either 4-OH-CB107 or Aroclor 1254 exposure (Table 1). In all groups, vaginal opening occurred at approximately PND 3436 (811 litters). Male preputial separation was completed at PND 4344 in all treatment groups.
Organ Weights of Dams (P0) and Neonates (F1)
At PND 21, maternal body weights and absolute or relative weights of the collected organs (liver, kidneys, adrenals, thymus, brain, spleen, uterus, ovary) of treated animals showed no differences compared to the control group. Absolute and relative liver weights from male and female offspring at PND 4 were significantly increased after exposure to Aroclor 1254 (44% and 38%, respectively, for relative liver weights; Table 2). Relative thymus weight at PND 4 was significantly reduced in both male (23%) and female (27%) offspring exposed to Aroclor 1254 in utero.
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At 11 months of age, plasma estradiol concentrations in female F1 offspring in the proestrous stage (determined by the appearance of a swollen uterus at necropsy) were significantly increased in the 5 mg/kg 4-OH-CB107 treatment group by approximately 230% compared to control animals (Fig. 3). Estradiol concentrations of female F1 offspring in which the uterus was not swollen showed no significant differences.
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DISCUSSION |
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The most pronounced developmental effect observed following exposure to 4-OH-CB107 was a striking and dose related prolongation of the estrous cycle in female offspring, measured between PND 210 and PND 231. The estrous cycle was monitored at this age in order to select the appropriate animals for the behavioral studies (described in Meerts et al., 2004). Because differences in the estrous cycle were noticed between the different treatment groups, a thorough investigation of the estrous cycle was performed. Although no data are available about the estrous cyclicity during the life cycle of the female animals, the differences observed at the age of 210231 days between the different 4-OH-CB107 treatment groups are very pronounced. The prolongation in the estrous cycle was also observed in the Aroclor 1254 treated animals, but it was less pronounced. The total length of the estrous cycle was significantly increased in female offspring exposed to 5 mg 4-OH-CB107. It is known that a hypothyroid state is associated with a higher incidence of pseudopregnancy (Tohei et al., 2000
). However, it is unlikely that the increase in the estrous cycle length in the current study is caused by hypothyroidism, since the prolongation of the estrous cycle was observed in offspring at the age of 11 months, and these animals did not show differences in thyroid hormone or TSH levels. Decreases in thyroid hormone levels (TT4 levels) following in utero exposure to 4-OH-CB107 were only visible in fetuses (Meerts et al., 2002
) and neonates at PND 4 (Meerts et al., 2004
).
Aroclor 1254 and aryl hardrocarbon (Ah) receptor binding PCB congeners have been reported to induce several adverse effects on mammalian endocrine function. For example, prolongation of the estrous cycle (by an increasing length of the diestrous stage) and a delay in the first estrous was observed in female rats after transplacental and translactational exposure to 30 mg/kg Aroclor 1254 for one month (Brezner et al., 1984). The increased length of the estrous cycle in female offspring in our study was also a result of a prolonged diestrous stage, determined by the appearance of large amounts of leucocytes in the vaginal smears. A stage representing 1120 days of diestrous is known as pseudopregnancy (De Feo, 1967
). In the present study, the total length of the diestrous stage did not exceed 7 days. In addition, the prolongation of the diestrous stage had no effects on the fertility of the females in this study. However, the disturbances in estrous cycle length may indicate that females exposed in utero to 4-OH-CB107 may show signs of reproductive senescence at an earlier stage in life compared to corn oil treated females. The first stage of reproductive senescence in rodents is an increase in mean cycle length. Most aging rats then enter a stage of persistent vaginal cornification (PVC), which is often followed by a repetitive pseudopregnancy and finally persistent anestrous (Finch et al., 1984
).
Strikingly, plasma estradiol (E2) levels in 11-month old female offspring were significantly increased by 230% in the 5 mg/kg 4-OH-CB107 exposed group (in the proestrous stage). High plasma E2 values are often observed in aging rats with a prolonged estrous cycle (Lu et al., 1994). It is demonstrated that higher amounts of plasma E2 in middle-aged rats during successive estrous cycles gradually diminishes the neuroendocrine responsiveness to the positive feedback effect of E2 on luteinizing hormone (LH) secretion (LaPolt et al., 1988
). Another possible explanation may be an effect of OH-PCB-107 on estrogen sulfotransferase activity. Kester et al. (2000)
showed that OH-PCBs, including 4-OH-CB107, are extremely potent inhibitors of the human estrogen sulfotransferase (hEST) in vitro. In fact, 4-OH-CB107 was one of the strongest of the 32 tested compounds with an IC50 of 0.150.25 nM. Estrogen sulfation is a normal route of reversible inactivation of estradiol. As a result of the inhibition of estrogen sulfation, OH-PCBs may increase the bioavailability of E2 in target tissues, thereby exerting an indirect estrogenic effect or mimicking the increase in plasma E2 levels observed in aging female rats.
4-OH-CB107, used as a model PCB metabolite in this study, is also known to possess anti-estrogenic activity in vitro in HeLa cells, or in human T47D breast tumor cells transfected with an estrogen-responsive luciferase gene construct (Moore et al., 1997; Meerts, unpublished results). In addition, Moore et al. (1997)
showed that 4-OH-CB107 significantly displaced [3H]E2 from the rat uterine cytosolic estrogen receptor, though <50% displacement was observed at the highest concentration used (103 M). It is thus very unlikely that the observed increases in E2 in our study are caused by binding of the metabolite to the estrogen receptor.
Next to the above-mentioned effects of 4-OH-CB107 on the estrous cycle length and estradiol concentrations, all other developmental effects observed were caused by the parent compound (Aroclor 1254) only. These effects include a significant increase in the female anogenital distance/crown-rump length ratio (AGD/CRL-ratio), an indicator of circulating androgen concentrations over time, or of decreased androgen responsiveness. This may indicate a possible partial "masculinization" of female offspring by Aroclor 1254 treatment. In addition, exposure to 25 mg/kg Aroclor 1254 significantly accelerated eye opening in the offspring by one day. Similar effects have been observed using either TCDD (Gray et al., 1997; Theobald and Peterson, 1997
) or Aroclor 1254 (Goldey et al., 1995a
). The effect on eye opening is most likely caused by a direct effect of the compound used (i.e., PCBs or TCDD) and not by the accompanying hypothyroidism observed in treated offspring, because hypothyroidism is typically associated with a delay in this developmental landmark (Adams et al., 1989
; Goldey et al., 1995b
). From the present study, it can be concluded that accelerated eye opening is most likely an effect of parent PCB congeners, and not their metabolites.
In conclusion, maternal exposure to the PCB metabolite 4-OH-CB107 results in a significant increase of the estrous cycle length and increased estradiol/progesterone ratios. The effects of the PCB metabolite are sex-related, because no effects could be detected on male accessory sex organ weights or testosterone levels at postnatal days 310 to 325. The well-known developmental effects of Aroclor 1254 (accelerated eye opening in treated offspring, increased AGD/CRL-ratio in female offspring), also shown in this study, could not be observed in offspring exposed to 4-OH-CB107 only. The adverse effects of 4-OH-CB107 on neurotransmitter levels and brain development in rat offspring exposed in utero are published elsewhere (Meerts et al., 2004).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at NOTOX Safety & Environmental Research B.V., Hambakenwetering 7, 5231 DD s-Hertogenbosch, the Netherlands. Fax: + 31-736406799. E-mail: ilonka.meerts{at}notox.nl.
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