Developmental immunotoxicity has gained significant attention recently because the “fetal basis of adult disease” hypothesis proposes that many chronic diseases including autoimmune diseases seen during adult stage of life may result from prenatal exposure to environmental contaminants [1
]. Recent epidemiological and experimental studies have provided increasing evidence to support this hypothesis. Thus, it is critical to protect the fetus from environmental insults and dietary supplements with such properties would be an ideal choice that can be used during pregnancy. In the current study, we demonstrate for the first time that RES can neutralize the developmental immunotoxicity of TCDD as well as block the changes induced by TCDD in the thymus of pregnant mice. These assertions are based on the following data that we obtained from this study: 1) RES reversed the effects of TCDD on thymic atrophy in non-pregnant mice (), pregnant mice and their fetuses (), 2) TCDD-induced alterations in T-cell subpopulations and co-stimulatory molecules were significantly blocked by RES ( and ), 3) RES ameliorated TCDD-induced apoptosis in thymic T cells of naive mice (), pregnant mothers and their fetuses (), 4) TCDD caused major alterations in T cell differentiation in the fetus particularly decreasing the proportion of double-positive (DP) T cells and increasing the percentages of double-negative (DN) cells, thereby suggesting that TCDD was inhibiting the differentiation of DN T cells into DP T cells. RES was able to restore the T cell differentiation by promoting DP T cell differentiation (), and 5) RES inhibited TCDD-induced expression of CYP1A1 in both mothers and fetuses ().
A well established epidemiological finding that supports the “fetal basis of adult disease” stems from the prenatal exposure to diethylstilbestrol (DES), a synthetic estrogen that was used in an estimated 5–10 million Americans, between 1938–1975, to prevent miscarriages or premature deliveries. Exposure to DES has been associated with an increased risk for breast cancer in “DES mothers” and a life time risk of cervicovaginal cancers in “DES daughters” [28
]. Exposure to DES has also been linked to a wide range of abnormalities in DES sons and daughters including immune system disorders such as increased incidence of autoimmunity, cancer and certain infections [28
]. Furthermore, experimental studies from our laboratory demonstrated that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery [29
]. Also, we noted that TCDD alters the process of thymic selection, possibly by enhancing negative thymocyte selection, whereas at the same time allowing autoreactive T cells to escape deletion in the thymus and immigrate to the periphery [30
]. Similarly, using genetically predisposed mice, developmental exposure to TCDD was shown recently to alter humoral immune functions and exacerbate a type III hypersensitivity lupus-like autoimmune disease [4
]. Together, such studies suggest that exposure to TCDD during development may have a long lasting impact on the immune functions leading to altered susceptibility to infections, autoimmune disease, and hypersensitivity reactions.
Previous studies from our laboratory have demonstrated that TCDD-induced thymic atrophy in the adult and fetus may result, at least in part, from induction of apoptosis [16
]. TCDD triggered the expression of several apoptotic genes, including Fas and FasL involved in the extrinsic pathway of apoptosis. We noted the presence of a dioxin response element (DRE) and five nuclear factor-kappaB (NF-kappaB) motifs on Fas promoter, and no DRE but two NF-kappaB motifs on FasL promoter [31
]. Further studies revealed that TCDD regulates Fas and FasL promoters through DRE and/or NF-kappaB motifs via activation of AhR [31
]. We have also demonstrated that apoptotic thymocytes including those exposed to TCDD, on one hand, upregulate the expression of CD3, αβTCR, IL-2R, and CD44 markers while down-regulating the expression of CD4, CD8, and J11d markers [16
]. Based on the ability of RES to inhibit CYP1A1 induction by TCDD, we suggest that RES may act as an AhR antagonist in vivo
, thereby blocking TCDD-mediated alterations in the TCR and co-stimulatory molecules, T cell differentiation, apoptosis and thymic atrophy. These data are consistent with other reports demonstrating RES-mediated down-regulation of CYP1A1 expression in vitro
and in vivo
It should be noted that previous studies, including those from our laboratory, have suggested that RES can act as an AhR agonist/antagonist [13
]. This may depend on the dose of RES used and the target cells that may express varying levels of AhR. For example, we have noted that RES at higher concentrations acts as an AhR agonist and promotes apoptosis in activated T cells [17
], however, at lower concentrations, it acts as a an AhR antagonist and blocks TCDD-mediated apoptosis in activated T cells (unpublished data). Numerous in vitro
studies using different cell lines have also suggested the AhR antagonistic activity of RES. RES was shown to inhibit the CYP activity by competing for the substrate binding site [13
]. In both HepG2 cells [14
] and human mammary epithelial cells [34
], RES was shown to inhibit the induction of CYP1A1 expression by TCDD. RES acted as an AhR antagonist and inhibited the dioxin effects on bone formation in vitro
]. Similar to RES, alpha-naphthoflavone has also been shown to act as both AhR agonist and antagonist based on the concentration [35
While majority of the published studies have investigated the effect of RES on the induction of CYP1A1 produced by TCDD, two recent studies also evaluated the effect of RES to neutralize dioxin-induced toxicity in vivo
]. In one study, TCDD-induced wasting syndrome was alleviated by treating mice for 28 d with subcutaneous injection of RES. However, in this study, RES failed to alleviate TCDD-induced hepatomegaly and thymic atrophy [15
]. This may be because C57BL/6 mice were given a very high dose of TCDD (100 µg/kg body weight) while treating them with lower doses of RES (20 mg/kg), when compared to the current study. Also, Jang et al
recently reported that the pretreatment of pregnant mice with oral resveratrol (50 mg/kg body weight) significantly reduced the incidence of cleft palate and the severity of renal malformations in the pups caused by in utero
exposure to TCDD [36
]. In this study, however, the effect on the immune system was not analyzed.
It should be noted that for effective neutralization of TCDD-induced toxicity in vivo
, the dose and route of administration of RES may be critical. In the current study, we administered RES by oral route because purified form of RES is readily available in the market for human consumption in the form capsules. Also, oral route reflects the natural dietary exposure to RES. However, RES given orally shows poor bioavailability due to its high rate of degradation by metabolism [37
]. This may be the reason why in a previous study, RES administered orally was not as effective as subcutaneous injection [15
]. Bioavailability of RES in the mouse is also short (10–60 min) and varies upon dose quantity [38
]. Yu and co-workers reported that RES at a lower dose (20 mg/kg) when administered into mice by i.p. injection, was converted to resveratrol glucuronide and resveratrol sulfate metabolites within 15 min that was detectable in the serum [40
]. No RES or its metabolites were detected after 1 h in the serum. In another set of experiments, when 60 mg/kg resveratrol was administered i.g. into mice, the same metabolites were detected in the sera at 30 min. No trace of RES in sera was observed after 30 min but the metabolites of RES were still detectable after 3 h. Further studies are necessary to address if metabolites of RES can mediate some of the beneficial effects of RES.
In an earlier study, we noted that 100 mg/kg body weight dose of RES was necessary to prevent inflammation in experimental autoimmune encephalomyelitis [17
] as well as colitis models [41
]. In the current study, we observed that a similar dose of 100 mg/kg body weight effectively ameliorated TCDD-induced immunotoxicity. RES has been used at high concentrations such as 500, 1000, and 1500 mg/kg body weight for 10 days, to inhibit tumor growth in BALB/c mice in a dose-dependent manner [8
] or 100 mg/kg body weight to delay tumorigenesis in rats [42
]. It should be noted that the dose tested in the current study is feasible to achieve in humans because the human equivalent dose of 100 mg/kg in mouse is 486 mg, considering an average human weight of 60 kg. Currently, there are several nutraceutical companies selling purified resveratrol in 500-mg quantities in capsule form. Thus, the dose used in the current study reflects the potential pharmacological/dietary supplement dose that is currently available in the market.
In summary, the current study suggests that RES may serve as an effective AhR antagonist in vivo and therefore may be an attractive candidate to prevent immunotoxic effects induced by environmental contaminants during pregnancy, thereby protecting the fetus from deleterious effects.