Developmental immunotoxicity of dexamethasone: comparison of fetal versus adult exposures

Toxicology 194 (2003) 163–176

Developmental immunotoxicity of dexamethasone: comparison of fetal versus adult exposures Rodney R. Dietert∗ , Ji-Eun Lee1 , John Olsen, Kevin Fitch, James A. Marsh Department of Microbiology and Immunology, College of Veterinary Medicine, Veterinary Medical Center, C5-135 VMC, Tower Rd., Cornell University, Ithaca, NY 14853, USA Received 26 June 2003; accepted 31 July 2003

Abstract Dexamethasone-21 phosphate was administered (s.c.) to pregnant CD rats at days 6–21 of gestation (0, 0.0625, 0.125, 0.25, and 0.5 mg/kg/day) with identical exposure of non-pregnant adult females. Some reproductive (anogenital distance) and growth (body weight) measures of pups were altered. In the juvenile (5 weeks), the delayed type hypersensitivity response to KLH was significantly reduced at all doses examined and this pattern continued into adulthood (13 weeks). In contrast, the DTH response of adults exposed to DEX was unaltered even at the highest dose. Few DEX-induced changes were seen in offspring or adult blood parameters or in splenocytes analyzed for cell surface makers (by flow cytometry). The thymus of both exposed pups (both ages) and adults showed a marked reduction in the medulla/lobe area beginning with the 0.125 mg/kg/day DEX exposure level. Macrophage production of TNF and NO was only marginally affected as was splenocyte production of IL-4 and IFN-gamma. In contrast, pups assessed as juveniles were significantly depressed in splenic IL-2 and IL-10 production. DEX exposure altered serum antibody levels across age groups with an increase of KLH-specific IgG (beginning with the 0.0125 mg/kg/day dose) while total IgE was reduced. These results suggest that while DEX exposure produces some common alterations following in utero versus adult exposure, fetal exposure (even at the lowest doses tested) produces marked and persistent functional loss (DTH) not evident in exposed adults. Furthermore, there was no apparent advantage in delaying immune assessment until the offspring reached adulthood. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Dexamethasone; Developmental immunotoxicology; In utero exposure; Rats; Immune assessment

1. Introduction Dexamethasone (DEX) is a synthetic glucocorticoid (GC) used in the treatment of autoimmune and inflammatory diseases (Ando et al., 1996; Wenting-van Wijk ∗ Corresponding author. Tel.: +1-607-253-4015; fax: +1-607-253-3384. E-mail address: [email protected] (R.R. Dietert). 1 Present address: Chevron Phillips Chemical Company LP, 1001 Six Pines DR., Suite 4015, The Woodlands, TX 77380, USA.

et al., 1999) and in combination with other drugs for anticancer chemotherapy (Segren et al., 1999). DEX has also been administered to neonates to treat respiratory distress syndrome (Tsukahara et al., 1999) and to pregnant women for both antenatal therapy (Crane et al., 2003) and induction of parturition during labor (Ziaei et al., 2003). It also serves as a model to examine the impact of glucocorticoid hormones produced during stress responses on host development and function including immune status. Prenatal increases in GCs have been shown to modify the development

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of several organs (Seckl, 1998; Celsi et al., 1998). LaBorde et al. (1992) reported that during gestational exposure, the thymus, spleen, adrenals, lung and kidneys were very susceptible to DEX in comparison with other organs (brain, heart, testis and long bones). But some changes can occur even among these organs. For example, prenatal DEX exposure in rats leads to increased oxidative cell death among brain cells (Ahlbom et al., 2000) as well as altered monoamine metabolism (Muneoka et al., 1997). It also produces a persistent hyperglycemia in adult rats exposed to DEX in utero (Nyrienda et al., 2001). Ghosh et al. (2000) recently described a possible basis for life-stage-related differences in sensitivity to DEX-induced toxicity. They demonstrated that the negative feedback of the glucocorticoid receptor in adult rats limits their cellular sensitivity to DEX. In contrast, rat fetal cells are relatively susceptible to GCs since embryos appear to lack autoregulation of the GC receptor. GCs including DEX are known to be immunosuppressive and have been shown to cause significant changes in T cell development and function (Ashwell et al., 2000). One hallmark of GC-induced immunosuppression is the induction of apoptosis among CD4CD8 double positive thymocytes. Additionally, GCs seem to target IL-2 production (Daynes and Araneo, 1989) and to cause a skewing in Th1 versus Th2 cytokine production at least while DEX exposure is concurrent (Ramirez et al., 1996; Ashwell et al., 2000). GCs are thought to be critical factors capable of influencing thymocyte development and through some process, regulating T cell repertoire by altering TCR avidity thresholds used in T cell selection (Ashwell et al., 2000). Therefore, it is not surprising that exposure to prenatal DEX might influence not only the T cell repertoire but also the production of autoreactive clones and the risk of autoimmunity in the offspring (Ashwell et al., 2000; Bakker et al., 2000). Age of exposure to DEX appears to be an important factor in immune outcome. Coe et al. (1999) found that the capacity of neonatal T cells to respond to allogeneic antigens was influenced differentially depending upon the timing of gestational exposure to DEX. Exposure of Rhesus monkeys to DEX during early pregnancy elevated the neonatal T lymphocyte MLR responses while mid-late gestational exposure to DEX produced suppression of the response. The authors

suggested that critical windows exist during gestation when the same extrinsic events may lead to radically different outcomes. The present study examined the effects of DEX exposure in rats comparing gestational versus adult exposures. Offspring were assessed at two ages to evaluate the nature and persistence of DEX-immunotoxic changes compared against adult exposure-induced changes. The results are discussed in light of known GC-induced immunosuppression as well as the developmental immunotoxicity literature.

2. Methods 2.1. Animals Ten- to 12-week-old Sprague–Dawley rats (Crl: CD BR) were purchased from Charles River Breeding Labs., Raleigh, NC, USA. Animals were time-mated and arrived on gestational day 2. Certified Rodent Chow (Purina #5002) obtained from Old Mother Hubbard, Lowell, MA, USA, and water were fed ad libitum. Water intake was monitored for each dam every 48 h throughout the gestational exposure periods. A 12-h light/12-h dark cycle at 65–75 ◦ F and 40–60% humidity was utilized during the studies and animals were individually housed in polycarbonate cages. Body weights were recorded weekly. Protocols complied with NIH guidelines and were approved by the Cornell University Institutional Animal Care and Use Committee. 2.2. Reagents Dexamethasone 21-phosphate, o-phenylenediamine dihydrochloride (OPD), LPS and ConA were purchased from Sigma–Aldrich (St. Louis, MO, USA). Peroxidase-conjugated goat anti-rat IgG was obtained from Jackson Immunoresearch Laboratories Inc. (West Grove, PA, USA). Calbiochem (San Diego, CA, USA) supplied the KLH. ELISA kits for IFN-␥, IL-2, IL-4, IL-10, and TNF-␣ were purchased from Biosource International (Camarillo, CA, USA). Fetal bovine serum, RPMI 1640 medium and Penicillin–Streptomycin were purchased from Grand Island Biological Corporation (GIBCO), Grand Island, NY.

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2.3. Experimental design The studies were conducted in the CD-1 rat using 15 dams per treatment group. The 2-day time pregnant dams and young adult females (non-pregnant adults) were randomly assigned to either the control or DEX-test groups. Control animals received daily s.c. injections of saline on gestational days 6–21. DEX-treated groups received s.c. injections of DEX diluted in saline at doses of 0.0625, 0.125, 0.25, or 0.5 mg/kg/day. After parturition, the offspring were culled at 7 days of age to four females per litter. Offspring were weaned at 21 days of age and housed individually. Fifteen female animals (each offspring female derived from a different dam) from each treatment group were used for experimental analyses. Body weights were recorded weekly from birth and anogenital distance was measured in the pups at 1 and 21 days after birth. Beginning 30 days after birth, vaginal opening was assessed daily. Rats received a primary and secondary sensitization with Keyhole limpet hemocyanin (KLH) in a 200 ␮l volume of sterile water (5 mg/ml) administered in the caudal tail fold at 3 and 4 weeks of age (for juvenile assessment) or at 11 and 12 weeks of age for adult assessment (Exon et al., 1990). Non-pregnant adults were immunized at the same time as adult offspring. A week after the second sensitization, all groups received a challenge injection of heat-aggregated (80 ◦ C for 1 h) 20 mg/ml KLH in 0.1 ml of saline in the footpad. The control footpad received saline alone. The delayed-type hypersensitivity response (DTH) was measured after 24 h using spring-loaded calipers (Dyer, Model 304). Animals were sacrificed at 5 or 13 weeks old for offspring, and 13 weeks post-treatment for non-pregnant adults with blood samples drawn, tissues excised and body weights recorded. Offspring were assessed for immunotoxicity both as juveniles and adults. Two ages were employed for evaluation within this design since prior evidence from our laboratory (Bunn et al., 2001a) had suggested that such a comparison was beneficial.

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blood determinations, while serum was obtained for analysis of KLH antibody and total IgE. Total leukocyte counts were performed on whole blood diluted 1:20 with erythrocyte lysing buffer. Leukocytes were enumerated on a hemocytometer. Differential cell counts were performed on blood smears stained with a Diff-Quick Staining Set (Dade Behring AG, Dudingen, Switzerland). 2.5. Organ collection Thymuses and spleens were collected, trimmed of adherent fat, and weighed at time of sacrifice. 2.6. Splenocyte preparation Spleens were removed aseptically at the time of sacrifice and single cell suspensions were prepared by forcing the spleens through 400 ␮m sterile nylon mesh. Splenocytes were washed, then erythrocytes were lysed with a buffered solution (pH 7.2) of 0.15 M NH4 Cl, 1.0 mM KHCO3 and 0.1 mM EDTA. After washing with Hank’s Balanced Salt Solution (HBSS), cells were plated at a concentration of 3×106 cells/well for unseparated preparations and at 6 × 106 for adherent cell preparations in 24-well culture plates. For adherent cell preparations, protein content was analyzed in wells using the bicinchoninic acid (BCA) method (Pierce Biochemical, Rockford, IL, USA). Adherent cells were stimulated with either 0 or 1.0 ␮g/ml LPS. Unseparated splenocytes were stimulated with either 0 or 5 ␮g/ml ConA. 2.7. ELISA for cytokine analysis IL-2, IL-4, IL-10 and IFN-␥ were measured in 72-h unseparated splenocyte supernatants stimulated with 5 ␮g/ml ConA in RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS) (<0.06 EU/ml). TNF-␣ was measured in 24-h supernatants generated from adherent splenocytes exposed to 1.0 ␮g/ml LPS.

2.4. Blood collection

2.8. Antigen-specific antibody ELISA

Peripheral blood was obtained by cardiac puncture of offspring at the time of sacrifice for total serum IgE and anti-KLH IgG antibody determinations. Heparin was used as an anticoagulant for whole

IgG antibody against KLH antigen was measured by ELISA as previously described (Miller et al., 1998), similar to Exon and Talcott (1995). An initial screening assay was performed to determine the optimal

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dilution of the sera. Briefly, KLH antigen was bound to a 96-well microtiter plate and incubated overnight at 4 ◦ C. The following day serum samples were diluted 1/500, added to the plates, and incubated for 1 h at 37 ◦ C. After washing, peroxidase-labeled mouse anti-rat IgG was added and incubated for 1 h. OPD was used to develop the plates and the absorbance was read at 450 nm. Relative absorbances were compared with positive and negative control pooled samples. 2.9. Total IgE ELISA Serum total IgE was measured by a sandwich ELISA using the method supplied by PharMingen. Briefly, plates were coated with purified anti-rat IgE (PharMingen, San Diego, CA, USA) washed and blocked, and then sera were added. Biotinylated anti-rat IgE (PharMingen, San Diego, CA, USA) was used as the detection antibody. Purified rat IgE kappa (Serotec, Raleigh, NC, USA) was used as the standard. After incubation with substrate (OPD), plates were read using an ELISA reader (Biotek EL312). 2.10. Nitric oxide (NO) Adherent splenocytes were incubated for 24 h in RPMI 1640 medium without phenol red supplemented with 2% FBS (<0.06 EU/ml) and stimulated with 0 or 1.0 ␮g LPS. NO production was measured by the Griess reaction (Green et al., 1982), which assesses accumulation of nitrite. 2.11. Surface staining of rat splenocytes for flow cytometry Splenocytes were prepared from tissue as previously described. One million cells per sample and one million cells of pooled sample used as controls were pelleted in 1-ml centrifuge tubes at 350 × g for 4-min. Supernatant was aspirated off and the cells pre-incubated with 1-␮g of purified Rat Fc BlockTM (Pharmingen, San Diego, CA, USA) in 50-␮l wash buffer (PBS, 1%FBS, 0.05% sodium azide, pH 7.2). Cells were gently agitated and incubated at 4 ◦ C for 10-min. The expression of leukocyte surface markers was determined by direct three-color staining

with monoclonal antibodies for PE-conjugated CD3, Cyc-conjugated CD4, and FITC-conjugated CD8, and single-color staining for FITC-conjugated CD45, tissue macrophages (PE-conjugated HIS36), and FITC-conjugated NK cells (Pharmingen), adding 1-␮g antibody in 50-␮l wash buffer to cells already in blocking buffer. Pooled cells were stained with these single- and three-color antibodies as well as isotype-matched, non-specific control antibodies: FITC-conjugated IgG1, PE-conjugated IgG2a, PE-conjugated IgG3, and Cyc-conjugated IgG2a (Pharmingen). Resuspended cells were incubated at 4 ◦ C for 30-min. in the dark. Cells were washed 3× with wash buffer, resuspended in 400-␮l wash buffer, and analyzed on the same day on a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA). Pooled cells stained with control antibodies were used to set the background gates for single-stained sample analysis. Single-stained (CD3, CD4 or CD8) pooled cells were used to set electronic compensation for multi-color fluorescence staining. A total of 10 000 events were recorded per sample. 2.12. Rat thymus microscopy Thymuses were removed and placed in 10% buffered formalin for 3 weeks and then shifted to 70% ethyl alcohol. The tissues were then placed in a 12-station tissue processor with the final station being paraffin wax for infiltration. Once the tissues went through the tissue processor, they were placed and processed through the tissue wax embedder (Tissue-Tek II). Sections were cut to a 6 ␮m thickness, placed on glass slides, stained with Hematoxylin–Eosin stain and mounted with cover slips for analysis. Image Pro Plus, version 4.0 (Media Cybernetics, CA, USA) video imaging software was used with a Leitz microscope mounted video camera (Hitachi) and a computer/video hard card. This software and camera (Hitachi) together, were capable of performing necessary calibrations for image analysis. The processed thymuses were analyzed using the Leitz microscope with a 1.6× plain optic calibrated to millimeters. Four random thymic lobes were selected from each sample for analysis. Using a software-tracing feature, the outline of the thymic lobe was first traced providing a denominator (perimeter measurement in mm). The Medulary

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region of the same thymic lobe was traced and used as a numerator (the perimeter measurement in mm). With the two measurements, we divided the Medulary region perimeter by the measurement of the whole thymic lobe and came up with a ratio/index reflecting the medullary region compared to the entire lobe (medulla + cortex). This procedure was performed for all four lobes and an average taken. 2.13. Statistical analysis Data were analyzed by a General Linear Model analysis of variance (ANOVA), followed by Fisher’s Least Significant difference test (Minitab Statistical Software, Minitab Inc., State College, PA, USA) to allow a multiple comparisons test when significance was indicated by ANOVA. Results were considered significant with a probability level of P < 0.05.

3. Results 3.1. General toxicity Since prior experiments employing the specific duration (days 6–21 of gestation) of exposure to DEX in utero had not been reported in CD-1 rats in the literature, we performed a preliminary experiment to establish dose ranges for use in the larger study. However, the highest dose (0.5 mg/kg/day) of DEX employed proved to be fetotoxic. As a result, later juvenile and adult assessments were performed only on the control and three lowest DEX treatment groups for the offspring. However, results from non-pregnant adults included the control and all four DEX treatment doses.

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3.2. Pregnancy outcome Table 1 shows the results of pregnancy/birth outcome following in utero exposure of CD-1 dams to DEX. Gestation length was significantly elevated over control in the highest dose group. Litter size was also reduced at this dose. Still-births were increased at the highest dose and even in the 0.25 mg/kg/day dose group. Pup body weight was reduced significantly from control in all DEX exposure groups. In contrast, sex ratio was unaltered by any exposure level. 3.3. Reproductive indices As part of the assessment, reproductive indices were collected at birth and through early postnatal development. Anogenital distance (AGD, presented alone and in the context of body weight and cubic body weight) was determined at birth and at 21 days of age. The age of vaginal opening was also determined. Table 2 shows these results for female pups exposed to DEX in utero. Four pups per dam were assessed and total N numbers ranged from 37 to 60 per group. AGD is known to be a sensitive reproductive measure for developmental toxicity. At birth, AGD was significantly increased compared with the control in the three highest DEX exposure groups. However, when AGD was expressed as a ratio relative to body weight or cubic body weight, all doses of DEX produced significantly increased measures in a dose response manner. At 21 days of age, the AGD varied with DEX dose, but it was clear that only the highest dose of remaining animals (0.25 mg/kg/day) showed a difference versus the control group. In this case, the AGD alone was reduced compared to control value but was significantly

Table 1 Pregnancy outcome following fetal exposure to dexamethasone-21-phosphate (mean ± S.E.M.) Dexamethasone-21-phosphate (mg/kg/day) 0 Gestation length (days) Litter size Stillbirths/litter Sex ratio (female/male) Pup body weight (g)

21.7 11.7 0.1 0.49 6.8

0.0625 ± ± ± ± ±

0.1 a,b 0.4 a 0.1 a 0.03 0.1 a

21.4 11.8 0.2 0.49 5.8

± ± ± ± ±

0.125 0.2 a 1.1 a 0.1 a 0.04 0.1 b

21.7 12.3 0.1 0.51 5.3

± ± ± ± ±

0.25 0.2 a,b 0.3 a 0.1 a 0.03 0.1 c

22.0 10.7 1.1 0.53 4.6

0.5 ± ± ± ± ±

0.1 b,c 0.4 a 0.3 b 0.03 0.1 d

22.3 7.1 3.3 0.45 4.0

± ± ± ± ±

0.1 c 0.8 b 0.6 c 0.05 0.1 e

Means within a row with different letters (a, b, c, d, e) are significantly different at P < 0.05. Fifteen dams per group were used for the experiment.

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Table 2 Reproductive indices in female pups following fetal exposure to dexamethasone-21-phosphate (mean ± S.E.M.) Dexamethasone-21-phosphate (mg/kg/day) 0

0.0625

0.125

0.25

0.5

At birth AGD (mm) AGD/body weight (mm/g) AGD/body weight1/3 (mm/g1/3 )

3.67 ± 0.05 a 0.54 ± 0.01 a 1.94 ± 0.02 a

3.66 ± 0.07 a 0.64 ± 0.01 b 2.05 ± 0.04 b

3.85 ± 0.07 b 0.73 ± 0.02 c 2.21 ± 0.04 c

3.98 ± 0.05 b 0.87 ± 0.02 d 2.39 ± 0.03 d

3.98 ± 0.06 b 1.00 ± 0.02 e 2.51 ± 0.04 e

At 21 postnatal days AGD (mm) AGD/body weight (mm/g) AGD/body weight1/3 (mm/g1/3 )

13.81 ± 0.17 a 0.24 ± 0.00 a 3.59 ± 0.04

13.59 ± 0.21 a 0.26 ± 0.01 b 3.65 ± 0.04

13.58 ± 0.16 a 0.27 ± 0.01 b 3.65 ± 0.04

12.52 ± 0.21 b 0.30 ± 0.01 c 3.53 ± 0.05

Age of first vaginal Opening (days)

33.8 ± 0.2

34.3 ± 0.3

34.2 ± 0.4

34.7 ± 0.3

–a –a –a –a

Means within a row with different letters (a, b, c, d, e) are significantly different at P < 0.05. Four pups from each dam per group were used for the experiment. a No female pup was alive in the highest dexamethasone exposed group when AGD measurement and the examination of first vaginal opening began.

elevated when adjusted for body weight. However, the ratio index of AGD adjusted for cubic body weight for the DEX-treatment groups did not differ from that of the control group. The age of vaginal opening determination approached significance (P = 0.08). 3.4. General comparisons DEX treatment of both non-pregnant adults and in utero exposed offspring produced significant reductions in body weight. Table 3 shows the results for the exposed offspring measured as juveniles (at

5 weeks of age) and adults (at 13 weeks of age). Table 4 shows the results for the same parameters for the non-pregnant adults exposed to DEX. Even the lowest DEX exposure produced body weight reductions in both groups. Relative spleen weights and relative thymus weights are also shown for the offspring assayed as juveniles and adults (Table 3). The equivalent data for the non-pregnant adults exposed to DEX are shown in Table 4. No changes were found in either organ parameter for either group tested as adults at any dose examined. In comparison among different ages of assessment, offspring exposed to DEX had an

Table 3 Body weight and relative organ weight in female pups following fetal exposure to dexamethasone-21-phosphate (mean ± S.E.M.) Dexamethasone-21-phosphate (mg/kg/day) 0

0.0625

0.125

122.48 ± 3.06 a

112.05 ± 2.61 b

111.37 ± 2.71 b

96.11 ± 3.85 c

Relative organ weight (g/100 g body weight) Spleen 0.36 ± 0.01 Thymus 0.43 ± 0.01 a

0.42 ± 0.02 0.46 ± 0.02 a

0.41 ± 0.02 0.46 ± 0.01 a

0.42 ± 0.02 0.51 ± 0.02 b

267.25 ± 3.90 b,c

280.20 ± 7.35 b

258.20 ± 7.98 c

0.27 ± 0.01 0.17 ± 0.01

0.27 ± 0.01 0.18 ± 0.01

0.28 ± 0.01 0.19 ± 0.01

At 5-week-old Body weight (g)

At 13-week-old Body weight (g)

300.79 ± 5.91 a

Relative organ weight (g/100 g body weight) Spleen 0.26 ± 0.01 Thymus 0.16 ± 0.01

0.25

Means within a row with different letters (a, b, c) are significantly different at P < 0.05. Fifteen pups per group were used for the experiment.

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Table 4 Body weight and relative organ weight in 26-week-old female rats following adult exposure to dexamethasone-21-phosphate (mean±S.E.M.) Dexamethasone-21-phosphate (mg/kg/day)

Body weight (g)

0

0.0625

0.125

0.25

0.5

339.41 ± 4.79 a

311.37 ± 6.37 b

324.00 ± 8.38 a,b

317.24 ± 6.31 b

307.27 ± 6.01 b

0.24 ± 0.01 0.11 ± 0.01

0.22 ± 0.01 0.10 ± 0.01

0.23 ± 0.01 0.09 ± 0.01

0.23 ± 0.01 0.10 ± 0.01

Relative organ weight (g/100 g body weight) Spleen 0.22 ± 0.01 Thymus 0.10 ± 0.00

Means within a row with different letters (a, b) are significantly different at P < 0.05. Fifteen rats per group were used for the experiment.

increased relative thymus weight at the highest DEX dose in the 5-week offspring. However, there was no difference detected when this parameter was assessed in the adult. 3.5. DTH measurements Fig. 1 shows the results of the delayed type hypersensitivity reaction to the protein antigen, Keyhole Limpet Hemocyanin (KLH). Following challenge with heat-aggregated KLH, the reactions were significantly reduced in all DEX exposure groups compared with the control. (As previously indicated, animals from the highest exposure group were not available for subsequent immune analyses). This is consistent with prior observations that, in general, DTH quantitation is a sensitive measure of developmental immunotoxicity. DEX exposure in utero suppressed the response of adult offspring versus controls for all DEX doses tested (Fig. 1B). Some dose response trend appeared evident as the 0.125 mg/kg/day dose differed from both the control as well as the lowest dose of DEX examined (0.0625 mg/kg/day). In contrast, the non-pregnant adults exposed to DEX exhibited no change in DTH responses to KHL (Fig. 1C). 3.6. Cell populations Both the total leukocyte count (TLC) and differential counts were determined on blood samples taken at 5 and 13 weeks of age from the exposed offspring and from the DEX-exposed non-pregnant adults. No changes in TLCs were evident following DEX exposure at any age (data not shown). When differential leukocyte counts were compared among treatment groups from the 13-week-old offspring and

the non-pregnant adults, no differences were found based on DEX exposure (data not shown). Flow cytometry analysis used antibodies directed against various cell surface markers and was performed on splenocyte preparations derived from both 5-week and adult offspring as well as non-pregnant adults. The protocol followed closely the PharMingen technical directions. Samples were analyzed and compared for CD3, CD4, CD8, the CD4/8 ratio, CD45RA (Ox-33 antibody), His36 (macrophage subset) and NKR (NK cells) (data not shown). While some age-related differences were evident in comparing the cell population incidences for juveniles versus adults, there were no DEX treatment-associated differences among detectable cell populations. The only exception was in the juvenile splenocyte analysis on the first day of CD-4 comparisons. However, this observation was not repeated during a second day of CD-4 analysis. In summary, splenic cell populations exhibited little change for the doses examined when evaluated following the long post-exposure recovery period. 3.7. Cytokine and metabolite analysis Unseparated splenocyte cultures were stimulated with Con A (5 ␮g/ml) for quantitation of IL-2, IL-4, IL-10 and IFN-gamma production. For production of TNF-alpha and NO, adherent cells received LPS (1.0 ␮g/ml) and the supernatants collected to measure cytokines/metabolite concentrations. ELISAs were performed using Biosource Int. kits for rat IFN-gamma, IL-2, IL-4, IL-10 and TNF-alpha. Additionally, nitric oxide was evaluated by measuring nitrite accumulation using a Griess reaction methodology.

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a

1 0.8

b

0.6

b

IL-2 (pg/ml)

Induration difference

1.2

b

0.4 0.2 0

(A)

0

0.0625

0.125

b c

1.5

b,c

IL-2 (pg/ml)

Induration difference

a

1 0.5 0

(B)

0

0.0625

0.125

4000 3500 3000 2500 2000 1500 1000 500 0

b

c

0.0625

a

0.0625

1.5

IL-2 (pg/ml)

Induration difference

0.125

0.25

c

5000

1

4000

b

b

3000 2000

a,b

c

1000

0.5

0

0

(C)

0.25

b

a,b

6000

2

0.125

c

b

0

(B)

0.25

a

0

(A)

0.25

2.5 2

8000 7000 6000 5000 4000 3000 2000 1000 0

(C) 0

0.0625

0.125

0.25

0.5

Dexamethasone-21-phosphate (mg/kg/day)

Fig. 1. Delayed type-hypersensitivity (DTH) to KLH in 5-week-old (A), 13-week-old (B) or non-pregnant adult (C) female rats exposed to dexamethasone-21-phosphate on gestational days 6 through 21. The DTH response was measured 24 h after heat-aggregated KLH challenge, as the difference in induration between KLH-injected and saline-injected control foot pads. Values represent mean ± S.E.M. Means with non-overlapping letters are significantly different at P < 0.05. Fifteen rats per group were used for the experiment. Each rat was from a different dam.

In the case of IFN-gamma, no difference among groups was evident from either juvenile or adult offspring or from non-pregnant adults (data not shown). The juvenile animals exhibit a DEX-associated decline in splenocyte IL-2 production (Fig. 2). In contrast, no DEX-treatment-related effects were noted for IL-4 production (data not shown). As with IL-2, juvenile animals had a reduced IL-10 production (Fig. 3) after

0

0.0625

0.125

0.25

0.5

Dexamethasone-21-phosphate (mg/kg/day)

Fig. 2. Production of IL-2 from Con A stimulated splenocytes. Splenocytes were derived from 5-week-old female offspring (A), 13-week-old female offspring (B) and non-pregnant female adults (C). Analysis was performed by ELISA (Biosource Protocol). Values represent mean±S.E.M. Means with non-overlapping letters are significantly different at P < 0.05. Fifteen rats per group were used for the experiment. Each rat was from a different dam.

DEX exposure (at the lower two doses). By 13 weeks of age the difference was no longer significant. Adults exposed to DEX at these doses showed no change in IL-10 production by splenocytes. 3.8. Anti-KLH serum IgG titers Fig. 4 shows the result expressed as ODs for the anti-KLH-specific IgG antibodies measured using an ELISA. DEX exposure followed by the recovery

R.R. Dietert et al. / Toxicology 194 (2003) 163–176

700

a

a,b Anti KLH IgG (ODs)

IL-10 (pg/ml)

b

400

a,b

300 200 100 0 0.125

0.25

a

a

0

0.0625

0.5

0.125

0.25

b

0

0.0625

0.125

1.5 1

b a

a

0

0.0625

0.5 0

0.25

(B)

0.125

2

800 600 400 200 0 0

0.0625

0.125

0.25

0.5

Dexamethasone-21-phosphate (mg/kg/day)

Fig. 3. Production of IL-10 from Con A stimulated splenocytes. Cells derived from 5-week-old female offspring (A), 13-week-old female offspring (B) and non-pregnant female adults (C). Analysis was performed by ELISA (Biosource Protocol). Values represent mean±S.E.M. Means with non-overlapping letters are significantly different at P < 0.05. Fifteen rats per group were used for the experiment. Each rat was from a different dam.

period of 5 or 13 weeks produced an elevation in antigen-specific IgG. In the 5-week-old offspring, both the 0.125 and 0.25 mg/kg/day groups showed a significant elevation over the control. At 13 weeks, the data were very similar. Titers were slightly higher than those seen at 5 weeks of age. The two highest treatment groups differed from the control and lowest treatment group but not from each other. The results in the non-pregnant adult were virtually identical to those in the offspring. Titers were in the same range as those seen at 5 weeks of age

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Dexamethasone-21-phosphate (mg/kg/day) Fig. 4. Rat serum IgG antibodies specific for KLH. Anti-KLH serum IgG titers were evaluated by sandwich ELISA and are presented as ODs for 5-week-old female offspring (A), 13-week-old female offspring (B), and non-pregnant female adults (C). Values represent mean ± S.E.M. Means with non-overlapping letters are significantly different at P < 0.05. Fifteen rats per group were used for the experiment. Each rat was from a different dam.

and possibly slightly lower than the 13-week values. The 0.125 and 0.25 mg/kg/day groups were elevated in antigen-specific IgG over the control and lowest treatment groups in a dose dependent manner. The highest group (0.5 mg/kg/day) (for which there was no offspring equivalent due to embryonic toxicity) was elevated in titer over controls but was significantly lower compared against the 0.25 mg/kg/day treatment group titers.

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These data suggest that given the immunization regime we employed, antigen-specific serum IgG was elevated by exposure to DEX, but the effect was not dependent upon age of exposure (within the ages examined). 3.9. Total serum IgE

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Total serum IgE was measured in an ELISA and is shown in Fig. 5. Overall, IgE levels were lowest in the juveniles, intermediate in the non-pregnant adults and highest in the young adults. DEX-associated changes in total IgE were seen for all ages. The results were similar in that DEX exposure produced a decline in serum IgE. At 5 weeks of age, the 0.125 exposure group differed from the control and lowest dose group and the highest DEX group differed from all others (suggesting a possible dose response relationship). In the 13-week offspring, all treatment groups were lower in IgE levels than the control and the decline was dramatic. In the non-pregnant adult the lowest dose producing a decline in IgE was identical to that seen at 5 weeks (0.125 mg/kg/day of DEX). While statistically different, the level of IgE suppression seen in the non-pregnant adult was considerable less compared with that observed in the offspring. The highest DEX exposure in the adult remarkably produced an elevated IgE levels well above that of the control. We have no ready explanation for this observation beyond the fact that the 0.5 mg/kg/day dose in the adult did produce other ‘breaks’ in the dose response pattern for other parameters. For example, compare IgG titer results where lower DEX dosing groups were elevated in a dose response manner but the 0.5 group deviated from this pattern. This was also seen for some cytokines.

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3.10. Thymus medulla/lobe ratios

Fig. 5. Total serum IgE. Serum IgE was evaluated using a sandwich ELISA procedure and concentration was determined based on a standard curve (expressed as ng/ml). Titers from 5-week-old female offspring (A), 13-week-old female offspring (B), and non-pregnant female adults (C) are presented. Values represent mean ± S.E.M. Means with non-overlapping letters are significantly different at P < 0.05. Fifteen rats per group were used for the experiment. Each rat was from a different dam.

Histological analysis of the H and E stained sections focusing on the cortico-medulary areas was performed with the thymus samples from 5- to 13-week-old offspring and from non-pregnant adults. The results are presented in Fig. 6 and expressed as the ratio of the medulla/entire thymic lobe. DEX exposure produced a significant reduction in the medullary area of the thymus compared with the cortex in the juveniles. The lowest dose producing this change was 0.125 mg/kg/day of DEX. For this dose as well as

the 0.25 mg/kg/day dose, the reduction in the medulla compared with the overall lobe was dramatic (1/3 to 1/2). For this parameter, there was no apparent difference in developmental sensitivity (offspring versus adults) nor was there a difference in outcome for the offspring when measured at 5 weeks versus 13 weeks of post-exposure. However, given the developmental difference in DEX-influenced DTH function seen between offspring and adults, the T-dependent

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Fig. 6. Thymus medulla/lobe ratio. Staining of tissues was done with Harris’s hematoxylin and eosin. Image Pro software (Media Cybernetics, Silver Spring, MD, USA) was utilized for analysis of thymus sections. Seven to eight thymuses were analyzed per group and four lobes were analyzed per animal. Data are presented as the ratio of the periphery of the medulla (mm)/the periphery of the lobe (cortex + medulla) for 5-week-old female offspring (A), 13-week-old female offspring (B) and non-pregnant female adults (C). Values represent mean ± S.E.M. Means with non-overlapping letters are significantly different at P < 0.05.

functional ramifications of thymus alterations in the embryo-neonate versus the adult may not be identical.

4. Discussion Direct comparisons of sensitivity to immunotoxicants across life stages are relatively uncommon in

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the literature. However, those cases that have been examined suggest that both quantitative and even qualitative differences may occur in immune outcome following fetal versus adult exposures. Examples of developmental immunotoxicants examined to date include chlordane (Barnett et al., 1987), TCDD (Gehrs and Smialowicz, 1999; Fine et al., 1989), tributyltin oxide (Smialowicz et al., 1989), heptaclor (Smialowicz et al., 2001), methoxyclor (Chapin et al., 1997), benzo[a]pyrene (Holladay and Smith, 1995), T2 toxin (Holladay et al., 1993), aflatoxin B1 (Dietert et al., 1985; Nelson-Ortiz and Oureshi, 1992), DES (Luster et al., 1979; Ways et al., 1980) and Pb (Faith et al., 1979; Bunn et al., 2001b). In most instances where direct comparisons have been possible, the embryo or neonate has proved to be more sensitive to immunotoxic modulation than the adult. This has led to recent considerations of the most effective strategies to accurately predict immunotoxic risk across life stages (Holsapple, 2002; Luster et al., 2003). For the present study, it is important to note that all results were based on a 5- or 13-week post-exposure recovery period. This is distinct from many reports of DEX toxicity where assessment occurs either during or immediately after exposure. Hence, the DEX-related effects may not be identical with those seen where exposure is concurrent with immunization and/or assessment. In fact, the issue of reversibility following DEX exposure is significant. Bakker et al. (1995) demonstrated that while effects of DEX exposure on the mature immune and neurological systems may be reversible, the effects following fetal exposure have a totally different kinetics. Prenatal exposure caused thymic changes in cell populations that were not evident following adult exposure. In addition, rat fetuses exposed to DEX had a delayed neonatal migration of T cells to the spleen compared with adult recovery following DEX exposure. Using in vitro studies, Kavelaars et al. (1995) demonstrated that human neonatal peripheral blood mononuclear cells were much more sensitive to DEX than were equivalent adult cells. Suppression of proliferation in neonatal cells was thought to be associated with DEX-induced suppression of IL-2 production. In contrast, DEX suppression of adult cells was thought to involve another mechanism (Kavelaars et al., 1995). In the present study, DEX exposure produced several immune changes including some not restricted by

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the age of exposure. Non-pregnant adult female rats exposed to DEX experienced altered thymus architecture with altered IL-2 production, elevated specific IgG and reduced serum IgE. Body weight (reduced) and IL-2 (elevated in the adults) seemed to be most sensitive parameters to change followed by thymus ratios and antibody changes. In the female offspring, the most sensitive parameters (based on the lowest DEX dose producing an effect) were body weight, anogenital distance adjusted for body weight, IL-10 production and DTH response. This was followed by sensitivity in IL-2 production, thymus ratios and antibody levels. The greatest age-related change was clearly the severe suppression of the DTH response, an effect confined to the offspring at both ages of assessment. The reduction in IL-10 production was also confined to the offspring. One potential tenet within developmental immunotoxicology is the issue of whether there could be a bias toward Th1 suppression if toxicant exposure occurs during critical windows of immune development associated with the latter stages of gestation. This has been discussed in recent reviews and workshops (Peden, 2000; Dietert et al., 2002). The idea is based on the fact that Th1 immune capacity appears to develop later than the default Th2 function. Hence, any chemically-induced suppression or delayed development of Th1 capacity could result in a relative shift toward Th2 function in the juvenile or adult offspring. Examples of such immunotoxic alterations have been associated with early exposure to Pb (Chen et al., 1999), methoxychlor (Chapin et al., 1997) and TCDD (Gehrs and Smialowicz, 1999). In the case of DEX, some studies suggested that in the presence of DEX there was either a suppression of both Th1 and Th2 function or a shift toward Th2 function (Wu et al., 1991; Ramirez et al., 1996). However, these studies analyzed changes concomitant with DEX exposure. In the case of the present study, DTH function was reduced following gestational exposure. But it was not clear that any pervasive shift toward Th2 function had persisted. In fact, there was no detectable change in offspring IL-4 or IFN-gamma production by splenocytes. While IL-2 production was reduced, so was the production of IL-10 as well as total serum IgE. We would hypothesize that DEX exposure produced similar immune changes in the offspring as in the

adult, but that the status of the fetal versus adult T cell system was probably a significant factor in the differential outcomes. Presumably, the adult system could either withstand the thymus-related effects to a greater extent than the developing embryo-neonate, or the effects were transitory in adults but persistent following fetal exposure. To sort among these possibilities, it would be helpful to know whether the adults experienced a temporal decline in DTH function during active exposure to DEX at the doses employed. Regardless, these findings support the view that fetal versus adult exposure to immunotoxicants can lead to differential immunotoxic outcomes. Acknowledgements The authors thank Forrest Sanders, Eunice Kan and Liz Leong for their technical support and Drs. Irshad Hussain and Michael Piepenbrick for assistance with the manuscript. The assistance of Dr. Fred Quimby in providing consulting on thymus histology is gratefully appreciated. This research was supported by a grant from the American Chemistry Council. References Ahlbom, E., Godvadze, V., Chen, M., Celsi, G., Ceccatelli, S., 2000. Prenatal exposure to high levels of glucocorticoids increases the susceptibility of cerebellar granule cells to oxidative stress-induced cell death. Proc. Natl. Acad. Sci. USA 97 (26), 14726–14730. Ando, T., Suganuma, N., Furuhashi, M., Asada, Y., Kodo, I., Tomoda, Y., 1996. Successful glucocorticoid treatment for patients with abnormal autoimmunity on in vitro fertilization and embryo transfer. J. Assist. Reprod. Genet. 13, 776–781. Ashwell, J.D., Lu, F.W.M., Vacchio, M.S., 2000. Glucocorticoids in T cell development and function. Annu. Rev. Immunol. 18, 309–345. Bakker, J.M., Kavelaars, A., Kamphuis, P.J.G.H., Cobelens, P.M., Van Vugt, H.H., van Bel, F., Heijnen, C.J., 2000. Neonatal dexamethasone treatment increases susceptibility to experimental autoimmune disease in adult rats. J. Immunol. 165, 5932–5937. Bakker, J.M., Schmidt, E.D., Kroes, H., Kavelaars, A., Heijnen, C.J., Tilders, F.J., Rees, E.P., 1995. Effects of short-term dexamethasone treatment during pregnancy on the development of the immune system and the hypothalamo-pituitary axis in the rat. J. Neuroimmunol. 63, 183–191. Barnett, J.B., Soderberg, L.S.F., Menna, J.H., 1987. The effect of chlordane on the delayed hypersensitivity response of BALB/c mice. Toxicol. Lett. 25, 173–183.

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