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Stimulation of the Immune Response in B6C3F1 Mice By Genistein Is Affected By Exposure Duration, Gender, and Litter Order1

Posted on: Tuesday, 18 October 2005, 03:01 CDT

By Guo, Tai L; Chi, Rui Ping; Germolec, Dori R; White, Kimber L Jr

ABSTRACT

The objective of this study was to determine whether immune responses could be differentially modulated by the phytoestrogen genistein (GEN) in mice from the 1st and 2nd litters, and whether the effects were persistent or reversible. B6C3F1 mice were exposed to a control or GEN-containing diet at 25, 250, and 1250 g/g for the 1st litters, and 500 g/g for the 2nd litters from d 0 of gestation to postnatal day (PND) 22, and through feeding after weaning. At PND42, anti-CD3 antibody-stimulated splenic T-cell proliferation and the percentages of T cells were increased in mice from the 1st litters at 250 and 1250 g/g GEN but not from the 2nd litters. At PND84, the activity of IL-2-treated NK cells was significantly increased by GEN in mice from the 2nd litters but not from the 1st litters. The activity of cytotoxic T cells (CTLs) was also significantly increased by GEN in male mice from the 2nd litters. However, the increases in the CTL activity were not significant when the male mice were shifted from GEN-containing food to control food at PND22. Additionally, the increases in T-cell activities in female mice from the 1st litters and male mice from the 2nd litters were associated with a decrease in the percentage of CD4^sup +^CD25^sup +^ T regulatory cells. Overall, the results demonstrated that GEN could enhance the immune responses in mice from the 1st and 2nd litters; however, the effects varied depending on the exposure duration, gender, and litter order. J. Nutr. 135: 2449-2456, 2005.

KEY WORDS: * genistein * developmental exposure * immune stimulation and litter order

Genistein (GEN),3 a major isoflavone in most soy products, was shown to interact with estrogen receptors (ERs) in vivo. In vitro, it has also been utilized as a tyrosine kinase inhibitor (1,2). Despite the hypothesized beneficial effects of GEN (e.g., decreased incidences of some hormone-related cancers), there are concerns about the potential long-term effects of this compound on human health, especially that of infants and young children (3). Infants fed soy milk formulas have plasma isoflavone levels that are orders of magnitude higher than those of infants fed human or cow's milk (4). The possible long-term effects of these relatively high levels of phytoestrogens during infancy are unknown. Epidemiologic studies indicate increased use of asthma or allergy drugs in young adults who were fed soy formula during infancy compared with those who were fed cow's milk formula (5). Additionally, phytoestrogens have been detected in amniotic fluid (6-8), suggesting that in utero exposure also occurs.

Our previous studies demonstrated that exposure to GEN (0-20 mg/ kg) in adult female B6C3F1 mice for 28 d by gavage increased the activities of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells (9). Additionally, splenic T cell number was increased in male and female Sprague-Dawley rats when the rats were exposed to GEN during gestation and lactation by feeding the dams a GEN-containing diet (0-1250 g/g), and via the diet from postnatal day (PND) 22 to PND64 (10). Importantly, exposure of experimental animals to natural estrogens such as estradiol or synthetic estrogenic compounds such as diethylstilbestrol has also been associated with immunotoxic effects, particularly when administered perinatally during lymphoid organ organogenesis (11-13). These effects include thymic atrophy, myelotoxicity, and suppression of NK cell activity.

It was reported that immune responses such as the IgE hypersensitivity reaction are affected by birth order in humans. Matricardi et al. (14) showed that the prevalence of atopy is much higher among first-born children, and that there is a 3% decrease in prevalence for each additional younger sibling. A significant association between the proliferation of cord blood mononuclear cells to house dust mite and birth order was also reported (15). In addition, there is evidence that the time of exposure to GEN predetermines its biological effects. For example, mammary cancer chemoprevention was demonstrated after prepubertal and combined prepubertal and adult genistein treatments but not after prenatal or adult-only treatments in rats (16).

The objective of the present study was to further evaluate whether the effects of GEN on the immune system in B6C3F1 mice were consistent with its estrogenic properties. Depue et al. (17) suggested that the concentration of free estrogen may be higher in first pregnancies due to a deficit in sex steroid-binding globulin production. It was hypothesized that immune responses could be differentially modulated by GEN in male and female mice from the 1st and 2nd litters, and the effects might be persistent or reversible depending on the sex. In this study, we evaluated the effects of GEN on the immune responsiveness in the F^sub 1^ generation (B6C3F1) of female C57BL/6 male C3H mice. Exposure to GEN occurred through dams during gestation and lactation, and from feeding starting on the day of weaning (PND 22) until PND 42 or PND 84.

MATERIALS AND METHODS

Animals and treatments. Both female C57BL/6 and male C3H mice (8- 12 wk old) were obtained from Charles River Breeding Laboratories. To obtain 2nd litter B6C3F1 mice, time-mated primiparous C57BL/6 mice (male C3H mice) at gestation day (GD) 14 were purchased, and pups were removed from these mice after they gave birth. Two weeks later, time-mated C57BL/6 mice in their 2nd pregnancy were generated through housing 2 of these female C57BL/6 mice and 1 male C3H mice in 1 cage (plug date = GD 0), and the pups from these mice in their second pregnancy were considered the 2nd litters. Age-matched time pregnant C57BL/6 mice in their first pregnancy were generated similarly through housing 2 virgin female C57BL/6 mice and 1 male C3H mice in 1 cage, and the pups from these mice in their first pregnancy were considered the 1st litters. Pregnant mice were housed individually in standard plastic cages with hardwood chip bedding. The female mice were randomly assigned to different treatment groups on d 1 of each study, and each group consumed 1 of the treatment diets described below and water ad libitum for 1 wk before mating. Each treatment group consisted of 4-8 mice. The animal room was maintained within a temperature range of 22 to 25C and relative humidity of 50 20% with a 12-h light cycle (0700-1900 h). After parturition, the offspring were housed together with their respective dams, 1 litter/cage, until weaning on PND 22, at which time the offspring were housed up to 4 same-sex littermates per cage. To eliminate litter effect, one mouse from each litter for each sex was randomly selected for evaluation.

The diet in a powdered form (5K96, purchased from Purina Mills) is a natural ingredient diet, formulated to be used in experimental protocols in which dietary estrogenic activity is a concern. It meets the nutrient specifications as shown for NIH-31 in the 1996 update, except that casein replaced the protein contributed by soy and alfalfa, soy oil is replaced by corn oil, and the vitamin mix is adjusted for irradiation (18). The control diet was assayed for genistein and daidzein after hydrolysis of conjugates. The genistein and daidzein concentrations in this diet were determined by LC- electrospray-MS-MS to be ~0.5 g/g (19). GEN with purity > 99% was mixed into the standard 5K96 feed every 3 mo by the Diet Preparation Staff, Bionetics at the National Center for Toxicological Research; each dosed batch of feed was analyzed by the Division of Chemistry, and it was stable for at least 6 mo when stored refrigerated.

The dams consumed the 5K96 diet containing 0-1250 g/g GEN (Toronto Research Chemicals) starting from ~2 wk before mating to PND 22. One important modification in the experimental design for the 2nd litter study was that only 1 concentration of GEN was used. The rationale was that the concentration of 500 g/g was in the range (250-1250 g/g) in which most of effects were observed in studies of the 1st litter. The F^sub 1^ mice were exposed to GEN during gestation and lactation, and to GEN-containing feed from the day of weaning (PND22) to PND42 or PND84. On PND42 and PND84, mice were killed by CO2 inhalation, and the spleens and thymuses were collected for immunological evaluations. All animal procedures were conducted under an animal protocol approved by the VCU Institutional Animal Care and Use Committee (IACUC).

Determination the numbers of thymocytes and splenocytes. The quantification of immune cells was performed as previously described (20). Briefly, splenocytes and thymocytes were prepared by mashing the spleens and thymuses between 2 slides with frosted ends. After being washed, the cells were resuspended in an RPMI complete medium and counted using a Coulter Counter ZII with the RBC lysed using a ZAP-OGLOBIN II lytic reagent (Coulter).

Flow cytometric analysis of splenocytes. To determine the percentages of splenocyte subsets, the respective cell types were labeled with an appropriate monoclonal antibody (mAb), conjugated with a fluorescent molecule for visualization as previously described (20). All of the antibodies were obtained from BD PharMin\gen. For CD4^sup +^ cells, a phycoerythrin (PE)-conjugated mAb specific for the L3T4 cell surface protein was used, and for CD8^sup +^ cells, a fluorescein isothiocyanate (FITC)-conjugated mAb specific for the CD8a (Ly-2) marker was used. For NK cells, an FITC- conjugated anti-mouse CD3e mAb and a PE-labeled anti-NK1.1 antibody were used. Additionally, FITC anti-mouse CD25 and IgM, and PE anti- mouse CD3 were also used to label splenocytes. Isotype-matched irrelevant antibodies were used as controls. After the addition of the reagents, the cells were incubated at 4C in the dark for at least 30 min. After incubation, the cells were washed twice and propidium iodide (PI) was added as a viability stain. After incubation with PI for 5 min, the cells were washed and enumeration performed on a Becton Dickinson FACScan Flow Cytometer. Nonviable cells were eliminated through setting a live gate excluding red fluorescence emerging from PI; a forward scatter threshold was set high enough to eliminate RBC. For each sample, 5000 PI-negative events were counted.

Natural killer cell activity. Our previous study suggested that GEN affected the interleukin (IL)-2 augmented NK cell activity (9); thus, similar assays were performed in this study. The activity of NK cells was assayed as described using ^sup 51^Cr -YAC-1 cells (21).

Anti-CD3 antibody-mediated spleen T cell proliferation. The proliferation of splenocytes in the presence of anti-CD3 antibody was performed as previously described (9). The incorporation of ^sup 3^H-thymidine into the proliferating cells was used as the endpoint of the assay, and the data were expressed as kBq/2 10^sup 5^ cells.

CTL activity. The assay for CTL activity was performed as previously described using ^sup 51^Cr-labeled P815 cells (9). Controls for spontaneous and maximum release were generated by culturing labeled target cells in the presence of either E-MEM medium or 0.1% Triton X-100, respectively.

Statistical analysis. Data are expressed as means SEM. Results were tested for variance homogeneity using Bartlett's test. Homogeneous data were analyzed using 1-way ANOVA; when significant, Dunnett's t test was used to determine differences between the experimental and vehicle control group. For nonhomogeneous data, a nonparametric ANOVA was used; when significant, differences between the control and experimental groups were determined by the Wilcoxon Rank test. Jonckheere's test was used to determine an exposure level- related trend. Male and female mice were analyzed separately. Differences were considered significant at P ≤ 0.05.

RESULTS

GEN on the body weight and organ weights. Exposure to GEN significantly decreased terminal body weight in 1st litter males at concentrations ≥ 25 g/g and in 1st litter females at 25 and 1250 g/g at PND42 (Table 1). Terminal body weight was still decreased in adult (PND84) 1st litter male mice at concentrations of 250 and 1250 g/g and in female mice at 1250 g/g (Table 2). However, terminal body weight was not decreased in 2nd litter male and female mice at 500 g GEN/g at either PND42 or 84 (Tables 1 and 2).

Absolute spleen weight and thymus weight in either 1st or 2nd litter mice exposed to GEN from GD0 to PND42 were not affected (Table 1); however, there was a significant increase in relative spleen weight in male mice at both 250 and 1250 g/g and in female mice at 25 and 1250 g/g from the 1st but not from the 2nd litter (Table 1). At PND 42, an increase in relative thymus weight occurred in 1st litter male mice only at 250 and 1250 g/g (Table 1). At PND84, relative spleen weight was increased by exposure to GEN in 1st litter male mice at 250 and 1250 g/g, whereas it was decreased in 2nd litter male mice at 500 g/g, and these changes were associated with a corresponding alteration in absolute spleen weight (Table 2). Neither absolute nor relative spleen weights were affected in female mice from either the 1st or the 2nd litters at PND 84 (Table 2).

TABLE 1

Effect of genistein exposure from GD0 to PND42 on terminal body weight and organ weights in B6C3F1 mice1,2

GEN on the activation of T cells. The proliferative response of splenocytes was evaluated in the presence or absence of anti-CD3 antibody, a T-cell stimulator. At PND42, there was a dose-related increase in the anti-CD3 antibody-stimulated splenic T cell proliferation in both 1st litter male and female mice, and the changes were significant at 250 and 1250 g/g (Fig. 1A and B). Basal splenocyte proliferation (38.3 7.5 kBq/2 10^sup 5^ cells in the treatment group vs. 24.5 1.9 kBq/2 10^sup 5^ cells in the control group) increased significantly in males at 1250 g/g but not in females (Fig. 1A and B). However, neither the anti-CD3 antibody- stimulated nor the basal splenocyte proliferation was altered by GEN at 500 g/g in 2nd litter male and female mice (Fig. 1C and 1D). To determine whether the enhanced T-cell proliferation was due to a change in the percentage of T cells, a flow cytometric analysis of the T cell population was performed. The percentages of CD3^sup +^ T cells increased significantly in both 1st litter male (Fig. 2A) and female (Fig. 2B) mice at 250 and 1250 g/g GEN. However, neither the percentage of CD4^sup +^ T cells nor that of CD8^sup +^ T cells was affected by GEN at 500 g/g in 2nd litter male and female mice (data not shown).

TABLE 2

Effect of genistein exposure from GD0 to PND84 on terminal body weight and spleen weights in B6C3F1 mice1,2

At PND84, neither the anti-CD3 antibody-stimulated nor the basal splenocyte proliferation was altered by GEN at 25-1250 g/g in 1st litter male and female mice (data not shown). Although there was no change in the anti-CD3 antibody-stimulated splenic T cell proliferation in 2nd litter male and female mice, exposure to GEN produced an increase (33%) in the basal splenocyte proliferation in male mice at 500 g/g GEN. However, this increase was not significant (P = 0.13) when the male mice were shifted from GEN-containing food to control food after weaning at PND22 (data not shown). A flow cytometric analysis was performed to determine the percentages of splenic T cell populations at PND 84. Although the percentage of CD4^sup +^CD8^sup -^ T cells was decreased by GEN at 1250 g/g in 1st litter females, and that of CD4^sup -^CD8^sup +^ T cells was decreased at 250 g/g in 1st litter males, the CD3^sup +^ T cell population was not affected (Table 3). In 2nd litter males, exposure to GEN at 500 g/g significantly increased the percentages of CD3^sup +^IgM^sup -^ T cells and CD4^sup -^CD8^sup +^ T cells (Table 3). In contrast, the percentages of CD4^sup +^CD8^sup -^ T cells increased significantly in 2nd litter females. These increases were not significant (P = 0.10-0.22) when the mice were shifted from GEN- containing food to control food after weaning at PND22 (Table 3).

FIGURE 1 Effect of genistein on spleen cell proliferative response to anti-CD3 antibody stimulation in F^sub 1^ mice at PND42. (A) Male mice from the 1st litters; (B) female mice from the 1st litters; (C) male mice from the 2nd litters; and (D) female mice from the 2nd litters. Mice were exposed to genistein, and anti-CD3 antibody-mediated splenocyte proliferation determined as described without or with stimulation. The right-side bars in the control groups are the spleen cell proliferative responses to anti-CD3 antibody stimulation. Values are means SEM, n = 5-8. Asterisks indicate a difference from the control: *P ≤ 0.05; **P ≤ 0.01.

The activity of CTLs was also determined in 2nd litter B6C3F1 mice at PND84. This activity was significantly increased by GEN at 500 g/g at the effector:target ratios of 25:1, 12.5:1, 6.25:1, and 3.125:1 in 2nd litter males (Figure 3A). However, these increases were not significant (P = 0.14-0.28) when the male mice were shifted from GEN-containing food to control food after weaning (Fig. 3A). CTL activity was not affected in 2nd litter females at PND84 after GEN exposure from GD0 to PND84 (Fig. 3B) or from GD0 to PND22 (Fig. 3B).

GEN on NK cell activity. Overall, exposure to GEN had minimal effects on NK cell activity in both male and female mice from the 1st and 2nd litters at PND42, although there were some increases in the cytotoxic activity in the 250 g/g-concentration group in female mice from the 1st litters (Table 4).

At PND84, exposure to GEN did not increase IL-2-augmented NK cell activity in either 1st litter male or female mice (data not shown). In contrast, the activity of IL-2-augmented NK cells in the 2nd litter mice at PND84 was significantly increased by GEN at 500 g/g (Fig. 4A and 4B), and these increases remained significant when the mice were shifted from GEN-containing food to control food after weaning (Fig. 4C and 4D).

Flow cytometric analysis of splenic NK cells (CD3^sup - ^NK1.1^sup +^) revealed that exposure to GEN did not affect the percentages of NK cells in either male or female mice from the 1st litters at PND 84 (Table 3). The percentage of NK cells was not affected in 2nd litter males; however, the percentage of NK cells was increased in 2nd litter females, and this increase remained significant when the female mice were shifted from GEN-containing food to control food after weaning (Table 3).

GEN on the percentages of splenic CD4^sup +^CD25^sup +^ regulatory T cells. To explore the possible underlying mechanisms that GEN enhanced the immune responses, a flow cytometric analysis of T-regulatory cells (CD4^sup +^CD25^sup +^) was performed. Exposure to GEN from GD0 to PND42 at 1250 g/g significantly decreased the percentage of CD4^sup +^CD25^sup +^ T cells in 1st litter female mice but not in male mice (Fig. 5A and 5B). However, exposure to GEN at 500 g/g from GD0 to PND42 did not change the percentage of CD4 CD25 T cells in either male or female mice from the 2nd litters (Fig. 5C and 5D). Exposure to GEN from GD0 to PND84 at 500g/g produced a significant decrease in the percentage of splenic CD4^sup +^CD25^sup +^ T cells in 2nd litter male mice (Fig. 5E) but not in female mice (Fig. 5F); however, this decrease was not significant (P = 0.15) when the male mice were shifted from GEN- containing food to control food after weaning at PND22 (Fig. 5E).

DISCUSSION

Our previous studies provided evidence that immune responses were altered after oral exposure to GEN at physiologically relevant concentrations in experimental animals (9,10). In this study, we further examined the effects of dietary GEN on immune responses in both 1st and 2nd litter B6C3F1 mice. Exposure of F^sub 1^ generation mice was via gestation and lactation, and through feeding after weaning at PND22. Although both human and cow's milk contain low levels of isoflavones (22), isoflavones were identified in amniotic fluid, suggesting that they may cross the placental barrier (6-8). For a 25-g mouse consuming 2 g of diet/d, the concentrations of GEN at 25, 250, 500, and 1250 g/g are approximately equivalent to doses of 2, 20, 40, and 100 mg GEN/(kg . d), respectively. For a 4-mo-old infant who consumes soy formula as directed by the manufacturers, ~6- 9 mg/kg body weight of isoflavones can be achieved (23).

In this study, the terminal body weights were decreased by GEN, at least at concentrations ≥ 250 g/g, in both 1st litter male and female mice at PND42 and PND84. However, the terminal body weights in 2nd litter mice at PND42 and PND84 were not affected. In our previous studies, terminal body weights were also decreased in F^sub 1^ Sprague-Dawley rats born from primiparous dams at a GEN concentration of 1250 g/g (10); this might be related to a significant decrease in food intake by dams in this high GEN concentration group (24). However, food consumption did not change in pregnant mice at GEN concentrations ≤ 500 g/g in this study (data not shown). These observations suggest that exposure to GEN might be associated with a general toxicity in 1st litter mice but not in 2nd litter mice. Additionally, the relative weight of the spleen was increased by GEN in 1st litter mice at PND42 and PND84 except for the female mice at PND84; this was consistent with the findings that the relative weight of the spleen was also increased in GEN-exposed Sprague-Dawley rats born from primiparous dams when the rats were exposed to GEN-containing feed (0-1250 g/g) during gestation and lactation and from feeding from GD7 to PND64 (10). In contrast, spleen weight in male mice from the 2nd litters was decreased at PND84. This decrease might be due to a change in erythrocyte components because there was no significant change in the number of white blood cells in the spleen (data not shown).

FIGURE 2 Flow cytometric analysis of splenic T cell populations in F^sub 1^ mice at PND42. (A) The percentage of CD3^sup +^ T cells in male mice from the 1st litters; (B) the percentage of CD3^sup +^ T cells in female mice from the 1st litters. Values are means SEM, n = 6-8. Asterisks indicate a difference from the control: *P ≤ 0.05; **P ≤ 0.01.

TABLE 3

Effect of genistein exposure from GD0 to PND84 on the percentages of differential splenocytes in B6C3F1 mice1

FIGURE 3 Effect of genistein on splenic CTL activity in F^sub 1^ mice from the 2nd litters at PND84. Mice were exposed to control or genistein (500 g/g)-containing diet, and the activity of CTLs against P815 cells determined as described. (A) Male mice; (B) female mice. Values are means SEM, n = 6-8. * Different from the control, P ≤ 0.05.

Our previous studies demonstrated that exposure to GEN by gavage increased IL-2-augmented NK cell activity in adult female B6C3F1 mice (9). Interestingly, the activity of IL-2-activated NK cells in GEN-exposed 2nd litter male and female mice at PND84 also increased in this study. This observation also agrees with our previous report that dietary GEN exposure increased NK cell activity in F^sub 0^ Sprague-Dawley rats when the rats were exposed from GD7 to PND 51 (10). However, GEN had minimal effect on the activity of NK cells in 1st litter male and female mice at PND84, which was consistent with our earlier report that GEN had no significant effects on NK cell activity in male and female Sprague-Dawley rats born from primiparous dams when the rats were exposed to GEN from GD7 to PND64 (10). Although the exact litter information was not available on the adult female B6C3F1 mice and F^sub 0^ Sprague-Dawley rats used in our previous studies in which there was enhanced NK cell activity after GEN exposure (9,10), it was most likely that these animals were from 2nd litters and higher, considering that animal suppliers usually breed the dams multiple times to produce more pups.

It should be noted that the increases in NK activity at PND84 in 2nd litter mice were maintained even after the mice were shifted from the GEN diet to the control diet at PND22, suggesting that the effect of GEN on NK cells in 2nd litter mice at PND84 might be due to an imprinting mechanism that occurred during developmental exposure because there is evidence that dietary GEN exposure alters methylation patterns in the mouse genome (25). The expression of perform was shown to be under the regulation of DNA methylation and chromatin remodeling (26). Thus, in 2nd litter male mice, the increase in NK cell activity might have been due to a change in the perforin gene expression because there was no change in the percentage of NK cells associated with the increase in NK activity. In 2nd litter female mice, the increase in NK cell activity was associated with an increase in the percentage of NK cells. Thus, mechanisms such as inactivation of the cell cycle regulatory genes (e.g., p16INK4A, p15INK4B, p21Waf1/ Cip1, p27Kip1, and p73) by DNA methylation could be involved (27).

TABLE 4

Effect of genistein exposure from GD0 to PND42 on the activity of NK cells in 1st litter female B6C3F1 mice1,2

In contrast, NK activity in 1st and 2nd litter males was not affected by GEN at PND42. Although the NK cell activities in 1st litter female mice were slightly increased at PND42 at GEN concentration of 250 g/g, this increase was not dose related. The mechanism for an increased response only in the 250 g/g diet group of 1st litter females is currently unclear. It might be due to the antiestrogenic effect of GEN at that concentration; however, at a higher GEN concentration such as 1250 g/g, other effects of GEN such as tyrosine kinase inhibition might also be present (2). Additionally, exposure to GEN did not significantly alter NK cell activities in 2nd litter female mice at PND42. Therefore, GEN had minimal effect on NK cell activity at PND42 in both 1st and 2nd litter mice. In humans, the age of 18 y corresponds approximately to PND42 in mice (28). Thus, exposure to GEN seemed to have minimal effect on the NK cell activity at early ages.

FIGURE 4 Effect of genistein (500 g/g) on IL-2-augmented splenic NK cell activity in F^sub 1^ mice from the 2nd litters at PND84. (A) Male mice exposed to control diet, GEN-containing diet from GD0 to PND84, or GEN-containing diet from GD0 to PND22, and then shifted to control diet; (B) female mice exposed to control diet, GEN- containing diet from GD0 to PND84, or GEN-containing diet from GD0 to PND22, and then shifted to control diet. Values are means SEM, n = 6-8. Asterisks indicate a difference from the control: * P < 0.05; ** P < 0.01.

In this study, 1st litter male and female mice exhibited an enhanced anti-CD3 antibody-mediated splenocyte proliferation at PND42, which was due in part to an increase in the percentages of splenic T cells. These increases are also consistent with the observation in Sprague-Dawley rats that the numbers of T cells were increased by GEN when the rats were exposed to GEN from GD 7 to PND 64 (10). Although the anti-CD3 antibody-mediated splenocyte proliferation at PND84 was not significantly altered by GEN in either male or female mice from the 1st and 2nd litters, an increase in the percentage of CD3^sup +^ T cells and CD8^sup +^ T cells, and more importantly, an increase in the CTL activity occurred in 2nd litter male mice at PND84. Thus, the T-cell activities in male mice were modulated by GEN irrespective of litter number. However, the T- cell activities in female mice were modulated by GEN only when they were from the 1st litters.

FIGURE 5 Effect of genistein on the percentage of CD4^sup +^CD25^sup +^ regulatory T cells in F^sub 1^ mice. (A) Male mice from the 1st litters at PND42; (B) female mice from the 1st litters at PND42; (C) male mice from the 2nd litters at PND42; (D) female mice from the 2nd litters at PND42; (E) male mice from the 2nd litters at PND84; and (F) female mice from the 2nd litters at PND84. "GEN to Ctr" = mice were shifted from the GEN (500 g/g) diet to the control diet at PND22. Values are means SEM, n = 5-8. * Different from the control, P ≤ 0.05.

The CD4^sup +^CD25^sup +^ regulatory T cell was shown to suppress immune responses against foreign antigens and pathogens (29-33). Importantly, exposure to GEN produced a decrease in the percentage of CD4^sup +^CD25^sup +^ T regulatory cells in 1st litter female mice at PND42, the time at which enhanced anti-CD3 antibody- mediated proliferation was observed. A decrease in the percentage of CD4^sup +^CD25^sup +^ T cells was also observed in GEN-treated 2nd litter male mice at PND84. When the male mice were shifted from the GEN diet to the control diet at PND22, the percentage of CD4^sup +^CD25^sup +^ T regulatory cells returned to a level that was comparable to that of the control mice, and the increase in CTL activity was no longer significant. Thus, the effect of GEN on CD4^sup +^CD25^sup +^ regulatory T cells might be responsible in part for its stimulatory effect on T cells.

One of the important mechanisms by which GEN exerts its effect onmultiple organ systems is to interact with ERs and compete with estrogen for binding (2). There is evidence that both ERα and ERβ are expressed in NK cells; however, ERβ rather than ERα might be responsible for 17β-estradiol-induced suppression of NK activity (34). The expression of ERs was also reported in CD8^sup +^ T cells (12,35). In contrast to NK cells, ERα but not ERβ is required for 17β-estradiol- induced increases in interferon-γ expression and Th1 responses (36-38). In our studies, the activities of both T cells and NK cells, at least in 2nd litter male mice at PND84, were increased by GEN. It is conceivable that GEN increases NK activity by antagonizing estrogen's suppressive effect through ERβ because GEN has 7-20 times higher affinity for ERβ than ERα (39,40). In contrast, it is unlikely that GEN increases CTL activity by functioning as an estrogen agonist through ERa because GEN's affinity for ERa is low (39,40). However, it is possible that binding of GEN to ERβ would leave more free 17β-estradiol to interact with ERα, thus enhancing T cell activity.

The exact mechanism for differential immune stimulation by GEN in B6C3F1 mice from the 1st and 2nd litters after developmental and adult exposures is currently unknown. An increased level of estrogen was reported in first pregnancies (17,41), and this may be responsible in part for the differential immunomodulation in the 1st and 2nd litters. Additionally, there is evidence that the serum level of corticosterone, an immunosuppressive factor, is reduced after GEN administration in rats (42). In a first pregnancy, there is an increased level of cortisol in serum (41), which may be an alternative explanation for the differences in GEN-mediated immunomodulation in the 1st and 2nd litters. However, the mechanisms mentioned above do not fully explain the differential effects of GEN on NK activity and T cell activity in male and female mice from the 1st and 2nd litters.

In conclusion, our results demonstrated that the activities of both NK cells and T cells could be differentially modulated by GEN in male and female mice from the 1st and 2nd litters during adult and developmental exposures. Furthermore, these effects varied depending on exposure duration, gender, and litter order. GEN modulation of immune responses in animals might shed some light on the epidemiologic finding (5) that there is an increase in the use of asthma or allergy drugs in young adults who were fed soy formula during infancy compared with those fed cow's milk formula. Both NK and T cells contribute extensively to the persistence and progression of the disease in asthma and allergy (43,44). Additionally, it would be of value to further examine whether the potential of GEN to modulate host resistances to tumors is related to its immunomodulatory effect in different periods of life. Thus, further study examining the association of GEN exposure with the development of asthma, autoimmunity, and subsequent autoimmune diseases is warranted.

ACKNOWLEDGMENTS

The authors thank D. L. Musgrove, X. L. Zhang, and R. D. Brown for their technical help, and Connie Weis (Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR) for providing GEN feed.

0022-3166/05 $8.00 2005 American Society for Nutrition.

Manuscript received 1 March 2005. Initial review completed 24 April 2005. Revision accepted 11 July 2005.

1 Supported by funds provided by National Institutes of Health R21ES 012286, and in part by National Institute of Environmental Health Sciences contract N01-ES-05454.

3 Abbreviations used: CTL, cytotoxic T cell; ER, estrogen receptor; FITC, fluorescein isothiocyanate; GD, gestation day; GEN, genistein; IL, interleukin; mAb, monoclonal antibody; NK, natural killer; PE, phycoerythrin; PI, propidium iodide; PND, postnatal day.

LITERATURE CITED

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Tai L. Guo,2 Rui Ping Chi, Dori R. Germolec,* and Kimber L. White, Jr.

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298-0613 and * Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

2 To whom correspondence should be addressed. E-mail: tlguo@hsc.vcu.edu.

Copyright American Institute of Nutrition Oct 2005

Source: Journal of Nutrition, The

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