Our previous studies have provided evidence that the immune responses were altered following oral exposure to GEN at physiologically relevant concentrations in experimental animals (9
). In this study, we have further examined the effects of dietary GEN on the immune responses in both the first and second litter B6C3F1 mice. Exposure of F1
generation mice was gestational and lactational, and through feeding after weaning at PND22. Although both human and cow’s milk contain low levels of isoflavones (22
), isoflavones have been identified in amniotic fluid, suggesting they may pass placental barrier (6
). For a 25 g mouse consuming 2 g chow every day, the concentrations of GEN at 25, 250, 500 and 1250 μg/g are approximately equivalent to doses of 2, 20, 40 and 100 mg GEN/kg/day, respectively. For a 4-month-old infant who consumes soy formula as directed by the manufacturers, approximately 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 the 250 μg/g concentration and above, in both the first litter male and female mice at PND42 and PND84. However, no changes in terminal body weights were observed in the second litter mice at PND42 and PND84. In our previous studies, decreases in the terminal body weights were also observed in F1
Sprague-Dawley rats born from primiparous dams at 1250 μg/g (10
), which might be related to a significant decrease of food intake in this high GEN concentration group by dams (24
). However, there was no significant change in food consumption in pregnant mice at GEN levels of 500 μg/g or lower in this study (data not shown). These observations suggested that exposure to GEN might be associated with a general toxicity in the first litter mice but not in the second litter mice. Additionally, the relative weight of the spleen was increased by GEN in the first litter mice at PND42 and PND84 except for the female mice at PND84, which was consistent with the findings that an increase in the relative weight of the spleen was also observed in GEN-exposed Sprague Dawley rats born from primiparous dams when the rats were exposed to GEN-containing feed (0 – 1250 μg/g) gestationally, lactationally and from feeding from GD7 to PND64 (10
). In contrast, there was a decrease in the spleen weight in male mice from the second litters 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).
Our previous studies have demonstrated that exposure to GEN by gavage increased IL-2-augmented NK cell activity in adult female B6C3F1 mice (9
). Interestingly, an increase in the activity of IL-2-activated NK cells in GEN-exposed second litter male and female mice at PND84 was also observed in this study. This observation is also in agreement with our previous report that dietary GEN exposure increased NK cell activity in F0
Sprague-Dawley rats when the animals were exposed from GD7 to postpartum day 51 (10
). However, GEN had minimal effect on the activity of NK cells in the first litter male and female mice at PND84, which was consistent with our previous 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 F0
Sprague-Dawley rats used in our previous studies in which enhanced NK cell activity was observed following GEN exposure (9
), it was most likely that these animals were from the second 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 the second litter mice were maintained even after the mice were shifted from GEN diet to control diet at PND22, which suggested that the effect of GEN on NK cells in the second litter mice at PND84 might be due to an imprinting mechanism occurred during developmental exposure since there is evidence that dietary GEN exposure alters methylation patterns in mouse genome (25
). The expression of perforin has been shown to be under the regulation of DNA methylation and chromatin remodeling (26
). Thus, in the second litter male mice, the increase in NK cell activity might be due to a change in the perforin gene expression because there was no change in the percentage of NK cells associated with the increase of NK activity. In the second litter female mice, the increase in NK cell activity was associated with an increase in the percentage of NK cells. Thus, the 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
In contrast, neither the NK activity in the first litter males nor that in the second litter males was affected by GEN at PND42. Although the NK cell activities in the first litter female mice were slightly increased at PND42 at GEN concentration of 250 μg/g, this increase was not dose-related. The mechanism for an increased response only at the 250 μg/g diet group in first litter females is currently unclear. It might be due to the anti-estrogenic effect of GEN at the 250 μg/g concentration; however, at a higher GEN concentration such as 1250 μg/g, other effects of GEN such as tyrosine kinase inhibition might also be present (2
). Additionally, exposure to GEN did not significantly alter the NK cell activities in the second litter female mice at PND42. Therefore, GEN had minimal effect on NK cell activity at PND42 in both the first and second litter mice. In human, the age of 18 years old is approximately corresponding to PND42 in mice (28
). Thus, exposure to GEN seemed to have minimal effect on the NK cell activity in early ages.
In this study, the first litter male and female mice exhibited an enhanced anti-CD3 antibody-mediated splenocyte proliferation at PND42, which was partially due 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 are increased by GEN when the animals 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 first and second litters, an increase in the percentage of CD3+
T cells and CD8+
T cells, and more importantly, an increase in the CTL activity were observed in the second litter male mice at PND84. Thus, the T-cell activities in male mice were modulated by GEN no matter whether the male mice were from the first or second litters. However, the T-cell activities in female mice were modulated by GEN only when the female mice were from the first litters.
regulatory T cell has been shown to suppress immune responses against foreign antigens and pathogens (29
). Importantly, exposure to GEN produced a decrease in the percentage of CD4+
T regulatory cells in the first litter female mice at PND42, the time when an enhanced anti-CD3 antibody-mediated proliferation was observed. A decrease in the percentage of CD4+
T cells was also observed in GEN-treated second litter male mice at PND84. When the male mice were shifted from GEN diet to control diet at PND22, the percentage of CD4+
T regulatory cells returned to a level that was comparable to the control mice, and the significance in the increase of CTL activity was also lost. Thus, the effect of GEN on CD4+
regulatory T cells might be partially responsible for GEN’s stimulatory effect on T cells.
One of the important mechanisms by which GEN exerts its effect on multiple organ systems is to interact with estrogen receptors and competing with estrogen for binding (2
). There is evidence that both ERα and ERβ are expressed in NK cells; but ERβ instead of ERα might be responsible for 17β-estradiol-induced suppression of NK activity (34
). The expression of ERs has also been reported in CD8+
T cells (12
). In contrast to NK cells, ERα but not ERβ is required for 17β-estradiol-induced increases in IFN-γ expression and Th1 responses (36
). In our studies, both the activities of T cells and NK cells, at least in second litter male mice at PND84, were increased by GEN. It is conceivable that GEN increases NK activity by antagonizing the estrogen’s suppressive effect through ERβ because GEN has 7–20 times higher affinity to ERβ than ERα (39
). In contrast, it is unlikely that GEN increases CTL activity by functioning as an estrogen agonist through ERα because GEN’s affinity for ERα is low (39
). However, it is possible that binding of GEN to ERβ would leave more free 17β-estradiol to interact with ERα, and thus, T cell activity is enhanced.
The exact mechanism for differential immune stimulation by GEN in B6C3F1 mice from the first and second litters following developmental and adult exposures is currently unknown. An increased level of estrogen has been reported in first pregnancy (17
), and this may partially be responsible for the differential immunomodulation in the first and second litters. Additionally, there is evidence that serum level of corticosterone, an immunosuppressive factor, is reduced after GEN administration in rats (42
). In first pregnancy, there is an increased level of cortisol in the serum (41
), which may be an alternative explanation for the differences in GEN-mediated immunomodulation in the first and second litters. However, the mechanisms mentioned above still could not fully explain the differential effects of GEN on NK activity and T cell activity in male and female mice from the first and second 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 first and second 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 epidemiological findings (5
) that there is an increase in the use of asthma or allergy drugs in young adults who were fed soy formula during infancy as compared to those who were fed cow milk formula. Both NK and T cells contribute significantly to the disease persistence and progression in asthma and allergy (43
). Additionally, it would be of value to further examine if the potential of GEN modulating host resistances to tumors is related to its immunomodulatory effect in different periods of life. Thus, further study to determine the susceptibility to develop asthma, autoimmunity, and subsequent autoimmune diseases following GEN exposure is warranted.