Reduced Levels of 1,25-Dihydroxyvitamin D3 in Rat Dams and Offspring after Exposure to a Reconstituted PCB Mixture

Hellmuth Lilienthal*,1, Annemarie Fastabend{dagger}, Jürgen Hany*, Hatice Kaya*, Astrid Roth-Härer*, Lothar Dunemann{dagger} and Gerhard Winneke*

* Departments of Neurobehavioral Toxicology and {dagger} Analytical Chemistry, Medical Institute of Environmental Hygiene, Auf'm Hennekamp 50, D-404225 Düsseldorf, Germany

Received January 10, 2000; accepted June 29, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies revealed effects of polychlorinated biphenyls (PCBs) and other polyhalogenated hydrocarbons on steroid hormone levels and hormone-dependent functions including behavior. In the present study serum concentrations of the vitamin D3 metabolites 25-hydroxycholecalciferol (25-D) and 1,25-dihydroxycholecalciferol (1,25-D) were determined in rat dams and offspring after exposure to a PCB mixture that was reconstituted according to the congener pattern found in human breast milk. Unmated females were exposed to diets adulterated with 0; 5; 20; or 40 mg PCBs/kg diet. Exposure started 50 days prior to mating and was terminated at birth. Gestational exposure reduced serum concentrations of 1,25-D in dams in a dose-dependent manner. Concentration of 25-D was also decreased at the time of delivery, but not at weaning. Determination of 1,25-D in offspring at weaning revealed reductions in both high-exposure groups. Levels of 25-D were diminished only at the highest exposure level. Internal PCB concentrations in adipose tissue and brains exhibited a linear relation to dosages in diet. Concentrations of PCBs in brains were similar in dams and offspring at birth, but decreased at the end of lactation in dams. In offspring, values increased during this period because of continued exposure via the milk. In the adipose tissue, PCB levels were much lower in offspring than in dams. To our knowledge, this is the first report of PCB-induced effects on vitamin D3 metabolites. In dams, reductions were seen even at the lowest exposure level used. Further studies are needed to evaluate the biological significance of these reductions in pregnant dams and possible consequences for the developing offspring.

Key Words: polychlorinated biphenyls; vitamin D; development; steroids; rat.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are a class of widespread environmental contaminants that accumulate in food chains, due to their chemical stability and lipophilicity. The biphenyl molecule can be substituted with chlorine at ten positions. Taking into account the substitution pattern and number of chlorine substituents, 209 different congeners may be formed, which were described by Ballschmitter and Zell (1980). Elevated exposure to PCBs has been detected in many mammals including man. It has been shown that PCBs are transferred to the fetus via the placenta and to the newborn organism via milk (Ando et al., 1986Go; Masuda et al., 1979Go; Takagi et al., 1986Go). Furthermore, a particular vulnerability to PCB exposure was reported in developing animals and human infants (Brouwer et al., 1995Go; Peterson et al., 1993Go; Schantz 1996Go).

In addition to effects on liver enzymes and on the nervous system, PCBs are known to interact with thyroid hormones and gonadal steroids (Brouwer et al., 1995Go; Safe 1995Go). For instance, the non-ortho-chlorinated, coplanar congener PCB 77 binds to the estrogen receptor in a competitive manner and can modulate ligand-regulated processes (Nesaretnam et al., 1996Go). Reductions in serum testosterone concentrations have been found in rats treated with another coplanar congener, PCB 169 (Yeowell et al., 1987Go). PCB 169 also impaired estradiol-induced up-regulation of uterine estrogen receptors as did the ortho-chlorinated compound PCB 153 (Patnode and Curtis, 1994Go). Technical mixtures of PCBs, which consist mainly of ortho-chlorinated congeners, are reported to increase the concentration of estrone sulfate in exposed pregnant guinea pigs (Lundkvist et al., 1987Go). Moreover, they reduce the binding of progesterone to its receptor in rabbit uterine mucosa (Lundholm, 1988Go), alter the activities of several testosterone hydroxylases (Haake-McMillan and Safe, 1991Go), and mimic the effects of estradiol on uterine weights and the gonadotropin response in anterior pituitary cells (Jansen et al., 1993Go). Hydroxylated metabolites of PCBs also bind to estrogen receptors (Connor et al., 1997Go; Korach et al., 1988Go; Ramamoorthy et al., 1997Go). Both PCB congeners and metabolites can produce either estrogenic or anti-estrogenic effects, depending on the test systems (Connor et al., 1997Go; Jansen et al., 1993Go).

Several steps of the synthesis and metabolism of steroids in general involve cytochrome P450-dependent enzymes (Vanden Bossche 1992Go). Since many isoforms of this group, including those that catalyze the metabolism of sex steroids, are influenced by PCBs (Haake-McMillan and Safe, 1991Go; Safe 1990Go, Yeowell et al., 1987Go, 1988Go), PCB-induced actions on steroids other than gonadal hormones are not unlikely. In the present study PCB-induced effects on levels of vitamin D3 metabolites were examined. Vitamin D3, or cholecalciferol, is a secosteroid, which means one of the rings in the steroid molecule is broken. It is converted into its active form, 1,25-(OH)2-cholecalciferol (1,25-D), in two hydroxylation steps, the first occurring in the liver and the second in the kidney (review in Kumar, 1991). Both steps are catalyzed by cytochrome P450-containing enzymes, the 25-hydroxylase (CYP27) and the 1{alpha}-hydroxylase, which has been referred to CYP27B1 (Jones et al., 1998Go).

The classical targets of 1,25-D are those directly related to calcium homeostasis in the body and comprise the enterocytes in the intestine, the osteoblasts in bone, and the distal tubule cells in the kidney. Recently, binding sites for 1,25-D were found in several non-classical target tissues, e.g. parathyroid glands, adrenals, pituitary, male and female gonads, islet cells in the pancreas, skin, heart, vascular and skeletal muscles, lung, liver, and brain (reviews in Bouillon et al., 1995; Bringhurst et al., 1998; Jones et al., 1998; Walters 1997). In addition, 1,25-D has immune effects (Hewison and O'Riordan, 1997Go). It regulates cell differentiation and proliferation and, consequently, exerts anti-tumor activity (Bouillon et al., 1995Go). Like other steroids, 1,25-D has genomic and non-genomic actions, the latter of which were studied particularly in the intestine, osteoblasts, muscles, and parathyroid (Bouillon et al., 1995Go). In myoblasts, a rapid activation of adenylate cyclase and protein kinase C was found (De Boland and Boland 1994Go).

PCBs have known effects on calcium homeostasis and transport in neurons (e.g., Kodavanti et al., 1998) and in microsomes of the sarcoplasmic reticulum (Wong and Pessah, 1996Go, 1997Go). Therefore, any change in concentrations of 1,25-D by PCBs may lead to disturbance of calcium balance resulting in reduced availability of calcium to cells; this may exacerbate effects on calcium-mediated processes.

Most studies have used either technical mixtures or single congeners to investigate PCB-induced effects. Neither approach accurately reflects environmental exposure of animals and humans, because the pattern of PCB congeners differs in technical mixtures and environmental samples (Safe 1994Go). Therefore, a mixture of PCBs reconstituted according to the pattern found in human milk was used in the present study. The overall PCB concentration in breast milk is highly correlated with the concentration in cord plasma and plasma values in children at the age of 42 months (Lanting et al., 1998Go), reflecting their PCB body burden. This analysis was based on four selected PCB congeners for which very similar patterns were reported in all three matrices. Since 1,25-D has been shown to be produced by rat placental tissue in vitro (Tanaka et al., 1979Go) and, in contrast to the adult rat, also by the fetal rat liver (Takeuchi et al., 1994Go), concentrations of 25-D and 1,25 were measured in PCB-treated rat dams and pups. In addition, measures of internal exposure, reproduction, and development of the offspring were included.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments
The measurements reported here are part of a study including several behavioral tests and analyses of sex steroids and neurotransmitters. For the whole study, we purchased 80 female and 40 male Long Evans rats (LE outbred, Møllegaard, Denmark), 4–5 weeks of age, with body weights of 60–70 g. After arrival in our laboratory, they were housed in group macrolone cages with a bedding of wood chips (4/cage). A 12-h light cycle (lights on from 0600 to 1800 h) was maintained. The room temperature was 23°C and the relative humidity was 55%. Food and water were available ad libitum. Four weeks after arrival, when the female rats weighed about 120 g, they were randomly assigned to 4 groups (20 females/group) which were exposed to 0, 5, 20, or 40 mg PCB/kg diet (Controls, RM05, RM20, and RM40 groups, respectively). These dosages result in an average daily intake of about 0, 0.5, 2.0, or 4.0 mg PCB/kg body weight (bw), respectively, for an average female rat with a bw of 200 g and a daily diet consumption of 20 g. Fifty days after the start of exposure females were mated with hitherto unexposed males for 10 days. Feeding of contaminated diets was continued throughout mating and gestation and was terminated at birth of the offspring, postnatal day 0 (PND 0). However, exposure of the offspring continued until weaning because of PCB transfer via the milk . All protocols involving animals were approved according to the German legal requirements for animal care in experiments.

On PND 0, litter sizes and weights were determined and the pups were examined for malformations. Measurement of pup weights was repeated on PND 5, 10, 15, and 21 when the offspring were weaned from their mothers. On PND 0, 3 dams/group were sacrificed with their litters. Brains and perirenal adipose tissue were removed for analyses of internal PCB concentrations, and blood samples were taken by heart puncture for determination of 25-D and 1,25-D. For analyses of PCBs, brains of all pups from a litter were pooled. Additional blood samples were collected from dams (5–7/group) and female offspring (7–8/group) on PND 21. In accordance with accepted standards (Holson and Pierce 1992), only one pup per litter was taken for collection of the samples. The number of blood samples for determination of vitamin D metabolites was limited since samples of other pups were needed for the analysis of sex steroids. For determination of internal exposure to PCBs on PND 21, 3 litters/group were used and brain and fat tissue samples of 2 female pups/litter were pooled for the analyses. Animals were randomly selected for all dissections. On PND 21, only female offspring were sacrificed, in order to save the males for behavioral studies.

PCB Mixture
The mixture of PCB congeners was reconstituted according to published results of several studies on the PCB pattern in human breast milk (Duarte-Davidson et al., 1992Go; Jensen 1991Go; Noren and Lunden 1991Go; Safe et al., 1985Go; Schulte and Malisch 1984Go). PCB congeners were obtained from Promochem (Wesel, Germany). They had a guaranteed purity of > 99.8% and were free of dibenzofurans. Congeners were selected that met the criteria of consistent detection in at least 3 of the 5 sources in a mean percentage of more than 2% of the whole PCB content. The mono-ortho-chlorinated congeners PCB 28 and PCB 105, as well as the di-ortho-chlorinated PCB 101 were also included, because they are also frequently and consistently detected in breast milk and adipose tissue in the reports given above. In addition, the coplanar congeners PCBs 77, 126, and 169 were added in quantities reported for human milk (Dewailly et al., 1991Go; Noren and Lunden 1991Go) to include congeners of all classes, coplanar, mono- and di-ortho-chlorinated PCBs. The composition of the resulting reconstituted mixture is shown in Table 1Go. The proportions of the congeners given in Table 1Go are the average values of the concentrations detected in the reports cited above.


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TABLE 1 Composition of the Reconstituted Mixture of PCBs
 
The reconstituted mixture was added to standard laboratory food pellets (Ssniff AM R-Z) by the manufacturer (Ssniff GmbH, Soest, Germany). The calcium content of the diet was 0.9% and the vitamin D3 concentration was 25 µg/kg diet. Diets were prepared once for the entire experiment. An independent laboratory (LUFA, Kiel, Germany) monitored the concentrations of PCBs in the diets. Deviations from the intended concentrations were < 2%. In the control diet, all PCB congeners were below the detection limit of 0.005 mg/kg diet.

Determination of 25-D and 1,25-D
Serum concentrations of 25-D and 1,25-D were determined in duplicate by standard commercial test kits (Immundiagnostik GmbH, Bensheim, Germany). For measurement of 25-D, samples were analyzed using a competitive protein-binding assay (CPBA), with vitamin D3 binding protein from goat serum and 3H-25-D as tracer after extraction with acetonitrile. Non-specific binding was 4.933% and the intraassay and interassay variations were 2.7% and 14.0%, respectively. The detection limit was 0.542 ng/ml. The level of 1,25-D was determined using a radio receptor assay (RRA), after extraction on a silica gel column (Extrelut-1) and separation from other vitamin D3 metabolites on a second silica gel column (Bakerbond spe). Non-specific binding was 7.5%, and the intra-assay and inter-assay variations were 4.2% and 15.0%, respectively. The detection limit was 1.135 pg/ml. Samples were analyzed in a blind fashion.

Analysis of PCB Tissue Concentrations
PCB exposure levels were analyzed in the complete brain, as well as in perirenal adipose tissue, by gas chromatography with electron capture detection (GC-ECD system, HRGC Mega 2, Fisons, Mainz, Germany). Briefly, after thawing, the brain samples were ground with a potter homogenizer (B. Braun, Melsungen, Germany). Adipose tissue and brains were vortexed with formic acid in a test tube. Afterwards, the PCBs were extracted by solvent extraction with n-heptane from the tissue-formic acid mixtures. The n-heptane extracts were purified by silica gel chromatography using petroleum ether as the mobile phase. The gas chromatographic separation of the PCBs was carried out on 2 capillary columns of different polarity (DB5, 30 m x 0.32 mm x 0.25 µm, and DB1701, 30 m x 0.32 mm x 0.25 µm; both from J and W Scientific, Köln, Germany). Further details concerning the analytical procedures will be published in a forthcoming paper. The 14 PCB congeners used to create the RM, and additionally PCB 52, were measured in the different specimens. Determination of PCB 52 was included since it is always measured in routine analysis by the laboratory. PCB detection limits in brain samples from PND 0 and PND 21 were between 0.005 and 0.01 mg/kg. The range of detection limits in adipose samples was 0.05–0.1 mg/kg (PND 0 samples) and 0.13–0.25 mg/kg (PND 21 samples). The precision was > 90% in all cases.

Statistical Analysis
Statistical analysis was conducted using the SAS statistical package (SAS Institute Inc., Cary, NC). Statistical analyses were litter-based. Reproductive measures like litter size and weight were evaluated by analysis of variance (ANOVA). Mortality rates were evaluated with the Chi2 test. Body weights of pups during the suckling period were analyzed by ANOVA, with repeated measures on the day factor. For post hoc comparisons, the REGWQ test (Ryan, Einot, Gabriel, Walsh multiple range test; SAS/STAT User's Guide, 1990) was calculated. The SAS statistical package was also used for fitting internal PCB concentrations to linear regression models. Because of the non-normal distribution of hormone values, the non-parametric Mann-Whitney U-test (2-tailed) was used for comparisons of groups (Siegel 1956Go). To test whether hormonal effects were dose-dependent, orthogonal trends were determined according to the non-parametric method of Marascuilo and McSweeney (Bortz et al., 1990Go). Probabilities of p < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Concentrations of Vitamin D3 Metabolites in Dams
Results for serum concentrations of 25-D and 1,25-D in dams on PND 0 and PND 21 are shown in Figure 1Go, upper and lower parts, respectively. On PND 0, concentrations of 25-D were reduced by about 20% in the RM05 group and by about 35% in both high-exposure groups in comparison to controls. These differences were significant (Mann-Whitney U-test, U = 0, p < 0.05, for each treated group vs. controls). Reductions of 1,25-D were also significant in all exposed groups when compared to controls (U = 0, p < 0.05 for each treated group vs. controls). The Marascuilo-McSweeney test revealed significant linear trends, indicating that effects were dose-dependent (Chi2 = 7.40, p < 0.01, and Chi2 = 15.23, p < 0.0005 for 25-D and 1,25-D, respectively). In the RM05 group, serum concentrations of 1,25-D were about 50% lower than control values, while in both higher exposure groups concentrations of 1,25-D were below the detection limit (< 1.135 pg/ml). Differences between RM05 rats vs. RM20 and RM40 rats were also significant (U = 0, p < 0.05 for RM20 and RM40 groups vs. the RM05 group).



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FIG. 1. Serum concentrations of vitamin D3 metabolites in dams at birth of the offspring (upper) and at weaning (lower); n.d., not detected; detection limit 1.135 pg/ml. There were significant dose-response relationships at both time points according to tests of the linear trend, p < 0.005; group comparisons: *significant vs. controls, p < 0.05.

 
On PND 21, no significant differences between groups were found for 25-D (p > 0.1 for all comparisons). However, as can be seen in Figure 1Go, there was a dose-dependent reduction of 1,25-D concentrations (linear trend: Chi2 = 9.58, p < 0.005). Concentrations were reduced by about 21%, 37%, and 74% in RM05, RM20, and RM40 rats, respectively. The differences of the RM40 group compared to controls and also to the RM05 group were significant (U-test, RM40 vs. controls: U = 2, p < 0.005; RM40 vs. RM05: U = 2, p < 0.05).

Concentrations of Vitamin D3 Metabolites in Offspring
Results for the serum concentrations of 25-D and 1,25-D in offspring are shown in Figure 2Go. At weaning, the RM40 group exhibited decreased serum levels of 25-D, which were 20–25% lower than values in all other groups. These differences were significant (U-test, RM40 vs. controls: U = 2, p < 0.01; RM40 vs. RM05: U = 11, p < 0.05; RM40 vs. RM20: U = 8, p < 0.05). The offspring of the RM40 group also had significantly reduced serum concentrations of 1,25-D (U-test, RM40 vs. controls: U = 1 2, p < 0.05; RM40 vs. RM05: U = 11, p < 0.05; RM40 vs. RM20: U = 11, p < 0.05). Percentages of decrease were about 15% and 33% in RM20 and RM40 rats, respectively, when compared to controls, while the RM05 offspring exhibited an increase of about 13% in comparison to controls. The linear trend describing the dose dependency of PCB-induced effects on 1,25-D in offspring was significant (Chi2 = 5.01, p < 0.05).



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FIG. 2. Serum concentrations of vitamin D3 metabolites in female offspring at weaning. The linear trend test was significant, p < 0.05; group comparisons: *significant vs. controls, p < 0.05.

 
Reproductive Measures
Effects on reproduction are shown in Table 2Go. The number of pups per litter was slightly decreased in the RM40 group in comparison to the other groups. However, this decrease did not reach statistical significance according to ANOVA [F(3,71) = 2.21, p < 0.1]. Litter weights were reduced by 13.6% and 19.5% in the RM40 group when compared to controls and RM05 rats, respectively. ANOVA revealed significant overall differences between groups [F(3,71) = 3.79, p < 0.05], which could be ascribed to the difference between the RM05 and the RM40 groups according to post hoc tests. Postnatal mortality between PND 0 and PND 15 was increased by a factor of about 2 in both high-exposure groups, but these differences were not statistically significant (Chi2 = 2.831, p < 0.1). Body weights of pups from birth until weaning were also decreased by PCB exposure (Table 3Go) according to ANOVA, with repeated measures on the day factor [F(3,53) = 4.14, p < 0.05], but the interaction between exposure and days was not significant (p > 0.1), indicating that the weight gain was not affected. Post hoc comparisons are given in Table 3Go.


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TABLE 2 Number of Litters and Number of Pups per Litter
 

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TABLE 3 Mean Pup Weights until Weaning
 
PCB Concentrations in Tissue
The sum of all PCB congeners measured in the adipose and brain tissue is shown in Tables 4 and 5GoGo. As expected, internal exposure increased in a dose-dependent fashion. In addition, this increase was linear in both brain and adipose tissue. Adjustment to linear regression models yielded F-values ranging from 33.48 to 1193.81 with corresponding error probabilities of p < 0.0001. Explained variances (r2 values) by dosage were 0.99 and 0.81 in brains of dams at PND 0 and PND 21, respectively. In the offspring the corresponding r2 values were 0.96 at birth and 0.76 at weaning. In the adipose tissue of dams the r2 values were 0.86 at PND 0 and 0.74 at PND 21. Despite the termination of PCB intake after birth, differences of concentrations in fat tissue of dams between PND 0 and PND 21 were only minor and not significant in both higher exposed groups. In the RM05-group values on PND 21 were even higher than values on PND 0. This increase was seen for all congeners measured. Corresponding concentrations in female offspring on PND21 were about 80% lower than the values in dams in the RM40 group.


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TABLE 4 PCB Concentrations in Adipose Tissue Controls000.00 ± 00.00000.00 ± 000.00Female offspring
 

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TABLE 5 PCB Concentrations in the Brain ControlsDams PND 0Offspring PND 0Dams PND 21Female offspring PND 21
 
In contrast, internal exposure in the brain of dams decreased between birth and weaning in all treated groups (Table 5Go). The offspring exhibited slightly reduced concentrations in comparison to their dams on PND 0, but concentrations increased during lactation. This resulted in higher PCB levels on PND 21 than on PND 0. Consequently, brain values in the offspring were about 4 times higher than values in dams at weaning.

PCB concentrations in the adipose tissue of dams were about 50 times higher than levels in the brain at birth, while at weaning, there was a factor of about 150 because of the decrease in PCB concentrations in the brain during lactation. In female offspring, the ratio of adipose tissue concentrations to brain concentrations was about 7 on PND 21.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to the reconstituted PCB mixture caused dose-dependent reductions in serum concentrations of 1,25-D in dams at birth and weaning of the offspring and also in the offspring at both high-exposure levels. Concentrations of 25-D were also diminished in dams at the time of delivery but not at weaning. In the offspring, there was a PCB-induced decrease in the group exposed to the highest dosage of PCBs. These results demonstrate that exposure to a breast milk-like mixture of PCBs leads to decreased concentrations of a hormone involved in calcium homeostasis. These decreases cannot be related to a reduced food intake, as deviations of food intake between dams of the different exposure groups were smaller than 5%. In addition, the concentrations of 25-D and, in particular, 1,25-D, are regulated (Jones et al., 1998Go). Thus, it is very unlikely that the observed hormonal reductions are due to an altered supply of the precursor vitamin D3 or calcium under normal conditions, e.g., on either vitamin D3-deficient or calcium-deficient diets. Since male pups were used in the behavioral part of our experiments, the reduction of 1,25-D in female offspring on PND 21—although before puberty—may be sex-related. Future studies should clarify if PCB-induced effects on vitamin D3 concentrations differ between sexes.

Previous work on PCB-induced effects in vitro and in vivo revealed exposure-related changes in intracellular calcium concentrations and calcium uptake by mitochondria and microsomes together with alterations in calcium-dependent signal transduction processes (Kodavanti et al., 1998Go, 1996Go, and 1993). Also, transient inhibition of excitatory postsynaptic potentials by PCB 52, which could be antagonized by the L-type calcium channel blocker nifedipine, was described in slices from the ventral hippocampus (Hong et al., 1998Go). Furthermore, ryanodine receptor-mediated calcium transport was altered by PCBs in microsomes from skeletal and cardiac muscles (Wong and Pessah, 1996Go, 1997Go). Similar influences on calcium homeostasis in liver, neural tissue, and heart have been found after treatment with 2,3,7,8-TCDD (Al-Bayati et al., 1988Go; Canga et al., 1988Go; Hanneman et al., 1996Go). This raises the question of whether PCB-induced reductions in 1,25-D serum concentrations contribute to disturbances in calcium homeostasis and calcium-dependent functions. While it is unlikely that 1,25-D is involved in the effects of PCBs in the in vitro systems mentioned above, the situation is different in vivo. An altered calcium balance due to reduced levels of 1,25-D in vivo may well modify PCB actions in the cellular systems which were examined in vitro, as decreases in 1,25-D may result in a reduced availability of calcium to these targets, thereby exacerbating the described effects. Further studies are needed to clarify PCB-induced effects on cellular systems after exposure in vivo and the consequences of reduced availability of calcium because of reduced 1,25-D concentrations.

In addition, it has been shown that 1,25-D, like other steroid hormones, has genomic and non-genomic effects (reviews, e.g., in DeBoland and Boland 1994; Bouillon et al., 1995; Norman 1997). Non-genomic, rapid actions have been found in several cell types involving voltage-gated Ca2+ channels (Caffrey and Farach-Carson 1989Go), direct interaction with PKC (Slater et al., 1995Go) or rapid changes in the intracellular location of PKC (Bhatia et al., 1996Go). Thus, rapid actions of 1,25-D involve processes similar to those which, as described above, have been shown to be affected by PCBs in other cell types. PCBs are also reported to influence functions and targets for which an action of 1,25-D is assumed. These include bone calcium metabolism and kidney function (Andrews 1989Go), testes (Hany et al., 1999Go), keratinocytes (Vos and Beems 1971Go), T lymphocytes (Tryphonas et al., 1989Go), insulin release (Fischer et al., 1996Go), adrenal medulla cells (Messeri et al., 1997Go), and skeletal muscles (Wong and Pessah 1996Go). Binding sites for 1,25-D were recently detected in many of these non-classical target tissues which, like the ovaries, depend directly on 1,25-D (Kwiecinski et al., 1989aGo), while in others, like the testes, substitution of calcium is sufficient (Kwiecinski et al., 1989bGo). Obviously, there are many parallels in targets for 1,25-D and PCBs, and it remains to be determined whether these parallels are merely due to a direct action of PCBs on calcium homeostasis as described in vitro, whether reduced levels of 1,25-D in vivo result in additional influences on calcium-dependent processes, or whether they even mediate some of the PCB effects. For instance, reductions in 1,25-D may be involved in the PCB-related hypotonicity observed in human children (Rogan et al., 1986Go), since skeletal muscle myopathies were found in 1,25-D–deficient humans and rats, which normalized after treatment with vitamin D (Walters 1997Go).

The calcium balance in vivo is of particular importance during pregnancy and development of the progeny. To meet the additional requirements for calcium in fetuses, there is an increase in calcium absorption in maternal intestine (Heaney and Skillman 1971Go) as well as in bone resorption in rats, sheep, and humans (Miller et al., 1982Go). In addition, the conversion of 25-D to 1,25-D is increased during gravidity in rats (Paulson et al., 1990Go) and sheep (Ross and Dorsey, 1991Go). Concentrations of 25-D and 1,25-D are lower in offspring than in dams in several species, and both steroids are transferred via the placenta; however, this transport appears to be small (Ross et al., 1990Go). In humans maternal and fetal levels of 1,25-D are correlated (Delvin et al., 1988Go), but correlations are otherwise weak because of the production of 1,25-D in the placenta (Tanaka et al., 1979Go), fetal kidney and liver (Takeuchi et al., 1994Go). It is likely, that PCBs, as they are transferred across the placental barrier, influence concentrations of 1,25-D in fetal tissues. Therefore, further measurements should examine possible reductions of 1,25-D levels in fetal matrices.

The consequences of a reduced supply with 25-D and 1,25-D during gestation and lactation are still uncertain. In mice lacking the receptor for 1,25-D impairments of development and growth were found only after weaning, even when weaning was delayed, suggesting the presence of a substituting factor in milk (Yoshizawa et al., 1997Go). In addition, vitamin D-deficient rats gave birth to normally developed offspring, although their reproduction rate was very low (Halloran and DeLuca (1979Go). Normal bone mineralization was found in rat pups born to vitamin D-deficient dams (Halloran and DeLuca, 1981Go) and transplacental gradients of calcium and phosphate were not affected by vitamin D deficiency (Brommage and DeLuca 1984Go). This suggests that pregnant rats can deplete their skeleton minerals sufficiently to adjust to the enhanced calcium requirements of the fetuses at the end of gestation (Care 1997Go). It is assumed that calcium transport across the placenta is mediated by parathyroid hormone-related protein (PTHrP) as this protein stimulates the transfer in sheep, while PTH itself is not effective (Rodda et al., 1989). In addition, in mice lacking the PTHrP gene, transplacental transport of calcium is insufficient (Kovacs et al., 1996Go), suggesting that normal fetal calcium supply is achieved by PTHrP and not by 1,25-D. On the other hand, congenital rickets has been described in human children (Moncrieff and Fadahunsi 1974Go), and decreased concentrations of 1,25-D in fetal blood are associated with reduced body size and low bone mineral content (Namgung et al., 1993Go). In rats, diminished growth of longitudinal bones was found in neonatal offspring born to vitamin D-deficient dams (Miller et al., 1983Go). Also, in 1,25-D receptor knockout mice the gene expression of calbindin-D9K was markedly decreased in the kidney and intestine at weaning (Yoshizawa et al., 1997Go). Under normal conditions 1,25-D causes an increase in this calcium-binding protein at the end of gestation in rat fetuses, resulting in an adult-like distribution pattern that most likely serves to prepare the offspring for the transition from placental transfer of calcium to intestinal absorption (Delorme et al., 1979Go). Thus, in the absence of gross abnormalities described in 1,25-D receptor null mutants, there may be subtle changes in developmental actions of 1,25-D at late gestation and during lactation. This is particularly so since interactions of 1,25-D with thyroid hormones and sex steroids have now been reported (Suarez et al., 1998Go; Walters 1997Go) that are known to exert influences on development (McEwen 1994Go; Porterfield and Hendrich 1994). Because of the incomplete and controversial data, the role of 1,25-D and the consequences of its lack during development is difficult to assess. Reduced levels of 1,25-D caused by exposure to PCBs and other chlorinated hydrocarbons may affect any process in which 1,25-D takes part. In this framework, it is noteworthy that hypomineralization in teeth of children correlated with PCDD and PCDF values in breast milk (Alaluusua et al., 1996Go). Also, the contribution of reduced levels of 25-D and 1,25-D to decreases in body weights at the highest exposure level and increases in mortality in both of the high-exposure groups seen in the present investigation remain to be elucidated.

In contrast to long-lasting effects of the reconstituted PCB mixture on testosterone concentrations in serum (Hany et al., 1999Go), effects on 25-D and 1,25-D levels appear to be transient, since pilot measurements of sera from adult males, which were littermates of the rats studied by Hany et al., indicated no differences between treatment groups (data not shown).

The internal PCB concentrations in adipose tissue and brain showed a linear relation to dosages in diets. Due to a technical failure, no fat concentrations could be determined in the RM05 and RM20 groups of the offspring. However, because of the linear relationship, one might expect values of about 7.5 and 30 µg/g in the RM05 and RM20 groups, respectively. PCB concentrations were comparable in brains of dams and offspring at birth, but decreased in dams during lactation, while levels in brains of the offspring increased in this phase because of the ongoing exposure by nursing. During lactation PCBs are mobilized from body stores of the dam and transferred to the offspring via milk. Since milk transfer exceeds the placental transfer in all species so far investigated by two to three orders of magnitude, this results in higher tissue concentrations in weanling pups than in fetuses (e.g. Masuda et al., 1979; Takagi et al., 1986; Montesissa et al., 1992; Vodicnik 1986).

In the present study, PCB values in the adipose tissue of dams were only slightly diminished during lactation. This suggests that during nursing, PCBs are mobilized first from other compartments than the adipose tissue in rats, which is supported by the decrease of PCB values detected in brains. Numerous studies in different species have reported that the depot in the adipose tissue is a deep compartment from which persistent PCB congeners are less rapidly removed than from several other soft tissues (e.g., Lutz et al., 1977; Morales et al., 1979). At the lowest exposure level used in the present study (RM05) even an increase was seen, which may be due to a redistribution of ingested PCBs after the termination of PCB feeding in this group. At weaning PCB concentrations in the adipose tissue were much lower in offspring than in dams, most likely because of the shorter duration of exposure.

It is remarkable that even at the lowest treatment level significant reductions of 1,25-D could be detected. The internal exposure levels in adipose tissue from dams of this group (RM05) are about 10–100 times higher than median values of current PCB concentrations in human populations (Brunn et al., 1990Go; Stellman et al., 1998Go). For persons at the upper tail of the distribution, these values decrease to 3–25.

In conclusion, exposure to a reconstituted PCB mixture which reflects the congener pattern of breast milk resulted in dose-dependent reductions in serum concentrations of the steroids 25-D and 1,25-D in rat dams and offspring. However, the biological significance of these decreases is uncertain and remains to be evaluated.


    ACKNOWLEDGMENTS
 
The authors wish to thank Hildegard Huss and Michael Lieverz for excellent technical help. Supported in part by the State of Baden-Württemberg, Germany, Program Environment and Health, grant PUG 97004 to H.L.


    NOTES
 
Part of the data were presented at the 40th Spring Meeting of the Deutsche Gesellschaft für Pharmakologie und Toxikologie, Mainz, Germany, 1999.

1 To whom correspondence should be addressed. Fax +49 211 3389 331. E-mail: lilien{at}rz.uni-duesseldorf.de. Back


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