Dietary approaches to positively influence fetal determinants of adult health

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Abstract
The aim of this study was to investigate whether dietary phase 2 protein inducers will decrease oxidative stress and associated hypertension and inflammation in female spontaneously hypertensive stroke-prone rats (SHRsp) and positively affect the health of their adult offspring. Phase 2 proteins decrease oxidative stress either by promoting scavenging of oxidants or decreasing the probability of oxidants being formed. Phase 2 protein genes are coordinately up-regulated by transcription factor heterodimers containing Nrf2 binding to the antioxidant response elements in the promoters. Sulforaphane, a metabolite of the glucosinolate glucoraphanin, is a potent inducer of phase 2 proteins. Broccoli sprouts high in glucoraphanin were used as the source of the dietary phase 2 protein inducers. From 5 wk of age, SHRsp were fed daily with either a defined diet (Grn−) or the diet plus dried broccoli sprouts containing 5.5 μmol sulforaphane-inducing equivalents (Grn+). Since glutathione (GSH) is central to many oxidant scavenging pathways, we examined, at 6 months of age, the GSH and oxidized-glutathione (GSSG) contents of the carotid artery, aorta, heart, and kidney. In all these tissues, the Grn+ diet increased GSH levels and decreased GSSG levels, indicating decreased oxidative stress. The activities of cytosolic glutathione peroxidase, the major peroxide scavenging enzyme, and glutathione reductase, which reduces GSSG to 2GSH were also increased in these tissues, again indicating decreased oxidative stress. Endothelial-produced nitric oxide (NO) is a major means of causing vascular smooth muscle relaxation. Increased production of superoxide anion plays a major role in the development of hypertension since superoxide combines with NO to form peroxynitrous acid, thereby decreasing NO diffusion to smooth muscle. NO is produced by endothelial nitric oxide synthase in response to elevations in intracellular Ca2+ generally due to activation of acetylcholine metabotropic receptors. At 6 months of age, portions of the thoracic aorta were used to measure endothelial function by suspending the aortic rings under a 1.0 g tension in a Kreb’s saline containing 1 μM indomethacin. Phenylephrine was used to induce contraction and in the stable tonic contraction state acetylcholine was introduced into the bath solution. Isometric tension was then measured. Relaxation expected for a normotensive rat would be ∼1.0 g. SHRsp on the Grn− diet had maximal relaxation of ∼0.3 g in response to acetylcholine (i.e., it is clearly impaired). SHRsp on a Grn+ diet had maximal relaxation of ∼0.6 g, a substantial improvement over the Grn− group but still not that of a normotensive rat. The data indicate that the Grn+ diet either facilitated NO diffusion from the endothelium to smooth muscle or that the smooth muscle was more responsive to NO. To delineate which, the relaxation of the aorta in response to the NO donor sodium nitroprusside (SNP) was examined. Animals on Grn− diet had a maximal response to increasing SNP concentrations of ∼0.6 g relaxation indicating that this group of animals had an impairment of smooth muscle response to NO. Animals on Grn+ diet had a significantly better response to SNP with maximal relaxation being ∼0.8 g. In toto, these data suggest that a Grn+ diet improved both endothelial function as well as the ability of vascular smooth muscle to respond to NO. The latter may be due to less peroxynitric acid formation and thereby less oxidative damage to vascular smooth muscle and, hence, better ability to respond to NO. The better endothelial function and better response of smooth muscle to NO correlated with significantly lower blood pressure (Fig. 1 ⤻ ). Oxidative stress activates the transcription factor NFκB, which then translocates to the nucleus promoting transcription of proinflammatory genes. We demonstrate that the decreased oxidative stress associated with a Grn+ diet is also associated with decreased activation of NFκB as determined by increased IκBα protein levels and decreased NFκB p65 protein levels (Fig. 2 ⤻ E). The decreased activation of NFκB was associated with lower levels of the proinflammatory protein inducible nitric oxide synthase (iNOS) in brain (Fig. 2A⤻ ) and kidney (Fig. 2C⤻ ) and less activation of microglia in the spinal cord. Pregnant females on Grn+ diet were maintained on this diet until offspring were weaned. These offspring were then placed on Grn− or Grn+ diet. Adult offspring on a Grn− diet had blood pressures significantly lower than the animals of the parental generation on a Grn− diet and identical to that of the parental generation on a Grn+ diet (Fig. 1)⤻ . Offspring placed on a Grn+ diet had significantly lower blood pressures than their siblings placed on Grn− diet. Compared with the parental generation on a Grn− diet, offspring of females on a Grn+ diet also had significantly less tissue inflammation as determined by brain (Fig. 2A⤻ ) and kidney iNOS (Fig. 2C⤻ ), kidney ICAM1 (Fig. 2B⤻ ) as well as 45 kDa tyrosine nitrosylated protein (Fig. 2D⤻ ), with offspring on a Grn+ diet having yet lower inflammation compared with their siblings on the Grn− diet. Another index of inflammation examined was the number of activated microglia in thoracic spinal cord. Here the parental generation on a Grn− diet had 22.8 ± 1.0 activated microglia/unit area; the parental generation on Grn+ diet had 16.7 ± 0.6 (P− diet...