Phthalates are high-production-volume synthetic chemicals with ubiquitous human exposures because of their use in plastics and other common consumer products. Recent epidemiologic evidence suggests that women have a unique exposure profile to phthalates, which raises concern about the potential health hazards posed by such exposures. Research in our laboratory examines how phthalates interact with the female reproductive system in animal models to provide insights into the potential health effects of these chemicals in women. Here we review our work and the work of others studying these mechanisms and propose a model for the ovarian action of di-(2-ethylhexyl) phthalate (DEHP). In vivo, DEHP (2 g/kg) causes decreased serum estradiol levels, prolonged estrous cycles, and no ovulations in adult, cycling rats. In vitro, monoethylhexyl phthalate (MEHP; the active metabolite of DEHP) decreases granulosa cell aromatase RNA message and protein levels in a dose-dependent manner. MEHP is unique among the phthalates in its suppression of aromatase and in its ability to activate peroxisome proliferator-activated receptors (PPARs). We hypothesize that MEHP activates the PPARs to suppress aromatase in the granulosa cell. MEHP-, PPAR alpha-, and PPAR gamma-specific ligands all similarly decreased estradiol production and RNA message levels of aromatase in vitro. Our model shows that MEHP acts on the granulosa cell by decreasing cAMP stimulated by follicle stimulating hormone and by activating the PPARs, which leads to decreased aromatase transcription. Thus, the environmental contaminant DEHP, through its metabolite MEHP, acts through a receptor-mediated signaling pathway to suppress estradiol production in the ovary, leading to anovulation.

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Soy phytoestrogens have been proposed as an alternative to estrogen replacement therapy and have demonstrated potential neuroprotective effects in the brain. We have shown that a high soy diet significantly reduces infarct size following permanent middle cerebral artery occlusion (MCAO). Here, we tested the hypothesis that a high soy diet would attenuate programmed cell death after stroke. Adult female Sprague-Dawley rats were ovariectomized and fed either an isoflavone-reduced diet (IFP) or a high soy diet (SP) for 2 weeks before undergoing 90 min of transient middle cerebral artery occlusion (tMCAO) followed by 22.5 h reperfusion. Infarct size, as assessed by triphenyltetrazolium chloride staining, was significantly reduced by a high soy diet (P<0.05). Apoptosis in the ischemic cortex, measured by TUNEL staining, was significantly reduced by the high soy diet. The number of active caspase-3 positive cells and caspase-mediated alpha-spectrin cleavage were also significantly decreased in the ischemic cortex of SP rats. Furthermore, nuclear translocation of apoptosis-inducing factor (AIF) was significantly reduced in the ischemic cortex of SP rats. Soy significantly increased bcl-x(L) mRNA and protein expression in the ischemic cortex compared with IFP rats. Immunohistochemistry revealed increased neuronal expression of bcl-2 and bcl-x(L) in the ischemic cortex of both IFP and SP rats following tMCAO. These results suggest that a high soy diet decreases both caspase-dependent and caspase-independent programmed cell death following tMCAO. Further, a high soy diet enhances expression of the cell survival factor bcl-x(L) following tMCAO, contributing to the neuroprotective effects of soy in the ischemic cortex.

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Estrogen is a powerful neuroprotective agent with the ability to induce trophic and antiapoptotic genes. However, concerns about negative overall health consequences of estrogen replacement after menopause have led to the adoption of other strategies to obtain estrogen's benefits in the brain, including the use of selective estrogen receptor modulators, high soy diets, or isoflavone supplements. This study sought to determine the ability of a high soy diet to induce neuroprotective gene expression in the female rat brain and compare the actions of soy with estrogen. Adult ovariectomized female rats were treated with 3 days of high dose estrogen or 2 weeks of a soy-free diet, a high soy diet, or chronic low dose estrogen. Different brain regions were microdissected and subjected to real time RT-PCR for neuroprotective genes previously shown to be estrogen-regulated. The principle findings are that a high soy diet led to the widespread increase in the mRNA for neurotropin receptors TrkA and p75-NTR, and the antiapoptotic Bcl-2 family member Bcl-X(L). Immunohistochemistry confirmed increases in both TrkA and Bcl-X(L). Chronic low dose estrogen mimicked some of these effects, but acute high dose estrogen did not. The effects of a high soy diet were particularly evident in the parietal cortex and hippocampus, two regions protected by estrogen in animal models of neurological disease and injury. These results suggest that a high soy diet may provide beneficial effects to the brain similar to low dose chronic estrogen treatment such as that used for postmenopausal hormone replacement.

Dietary soy and soy isoflavones are neuroprotective in experimental cerebral ischemia. Because these isoflavones have estrogenic properties, we hypothesized that, like estrogens, they would inhibit acute vascular injury and the detrimental acute increase in hypoxia-induced vascular endothelial growth factor (VEGF) that leads to cerebral edema after stroke. Mature ovariectomized female Sprague Dawley rats were fed soy-free or soy-containing diets for 4 weeks followed by 90 minutes of transient middle cerebral artery occlusion. Similar to estrogens, dietary soy significantly reduced cerebral edema and vascular apoptosis 24 hours after stroke. Soy also inhibited the ischemia-induced increase in cortical VEGF and VEGF receptor (VEGFR)-2 protein expression observed 4 and 24 hours after stroke, although mRNA levels increased. The reduction in VEGF/VEGFR-2 was associated both with decreases in receptor phosphorylation and signaling to AKT and endothelial nitric oxide synthase. Furthermore degradation of the VEGFR-2 was increased with dietary soy. The primary ischemic stimulus for VEGF, hypoxia-inducible factor 1α (HIF1α), was similarly reduced by dietary soy 4 hours after transient middle cerebral artery occlusion in both the cortex and striatum. The inhibition of HIF1α activity was further confirmed by a significant decrease in the HIF1α-activated apoptotic mediator BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Nip3-like protein X). These data suggest that soy isoflavones target events early in the ischemic cascade as part of their neuroprotective actions and counterbalance some of the detrimental effects of the endogenous response to cerebral injury.

High soy (HS) diets are neuroprotective and promote vascular dilatation in the periphery. We hypothesized that a HS diet would promote vascular dilatation in the cerebrovasculature by mimicking estradiol's actions on the endothelial nitric oxide synthase (eNOS) system including increasing eNOS expression and decreasing caveolin-1 expression to increase nitric oxide (NO) production. Ovariectomized rats were fed HS or a soy-free diet (SF) ± low physiological estradiol (E2) for 4 weeks. Neither E2 nor HS altered middle cerebral artery (MCA) structure or vascular responses to acetylcholine, serotonin, or phenylephrine. Estradiol enhanced bradykinin-induced relaxation in an eNOS-dependent manner. Although E2 and HS increased eNOS mRNA expression in the brain and cerebrovasculature, they had no effect on eNOS protein expression or phosphorylation in the MCA. However, E2 decreased caveolin-1 protein in the MCA. In MCAs neither E2 nor HS altered estrogen receptor (ER) alpha expression, but E2 did reduce ER beta levels. These data suggest that HS diets have no effect on vascular NO production, and that E2 may modulate basal NO production by reducing the expression of caveolin-1, an allosteric inhibitor of NOS activity. However, the effects of E2 and HS on the cerebrovasculature are small and may not underlie their protective actions in pathological states.