Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system — PNASPNAS 
Proceedings of the National Academy of Sciences of the United States of America 
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  Advertise Contact Feedback Site Map Expand+Dietary approach to attenuate 
oxidative stress, hypertension, and inflammation in the cardiovascular system
  Lingyun Wu*, M. Hossein Noyan Ashraf*, Marina Facci*, Rui Wang†, Phyllis G. 
  Paterson‡, Alison Ferrie§, and Bernhard H. J. Juurlink*,¶
+ Author Affiliations
  Departments of *Anatomy and Cell Biology and †Physiology, College of Medicine, 
  and ‡College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, 
  SK, Canada S7N 5E5; and §Plant Biotechnology Institute, National Research 
  Council, Saskatoon, SK, Canada SW9 OW9 
  Communicated by Paul Talalay, The Johns Hopkins University School of Medicine, 
  Baltimore, MD, March 22, 2004 (received for review October 17, 2003) 
 Next SectionAbstract
Imbalance between production and scavenging of superoxide anion results in 
hypertension by the inactivation of nitric oxide, and the increased oxidative 
stress from the resultant peroxynitrite that is produced promotes inflammatory 
processes such as atherosclerosis. Induction of phase 2 proteins promotes 
oxidant scavenging. We hypothesized that intake of dietary phase 2 protein 
inducers would ameliorate both hypertension and atherosclerotic changes in the 
spontaneously hypertensive stroke-prone rat. For 5 days/week for 14 weeks, we 
fed rats 200 mg/day of dried broccoli sprouts that contained glucoraphanin, 
which is metabolized into the phase 2 protein-inducer sulforaphane (Group A), 
sprouts in which most of the glucoraphanin was destroyed (Group B), or no 
sprouts (Group C). After 14 weeks of treatment, no significant differences were 
seen between rats in Groups B and C. Rats in Group A had significantly decreased 
oxidative stress in cardiovascular and kidney tissues, as shown by increased 
glutathione (GSH) content and decreased oxidized GSH, decreased protein 
nitrosylation, as well as increased GSH reductase and GSH peroxidase activities. 
Decreased oxidative stress correlated with better endothelial-dependent 
relaxation of the aorta and significantly lower (20 mm Hg) blood pressure. 
Tissues from Groups B and C had considerable numbers of infiltrating activated 
macrophages, indicative of inflammation, whereas animals in Group A had few 
detectable infiltrating macrophages. There is interest in dietary phase 2 
protein inducers as means of reducing cancer incidence. We conclude that a diet 
containing phase 2 protein inducers also reduces the risk of developing 
cardiovascular problems of hypertension and atherosclerosis. 
Oxidative stress has been implicated as a causal factor in diseases such as 
hypertension (1–3) and atherosclerosis (4). Increased production, or decreased 
scavenging, of oxidants such as superoxide anion can give rise to hypertension 
by superoxide interacting with nitric oxide, forming peroxynitrite (5–7), 
thereby decreasing nitric oxide availability for smooth muscle relaxation 
function. The peroxynitrite formed is a strong oxidant that promotes 
inflammatory processes by activating transcriptional factor complexes such as 
NF-κB (8). Such inflammatory processes result in the formation of 
atherosclerotic lesions (9). In principle, decreasing oxidative stress and 
associated inflammation should provide the beneficial effect of preventing 
and/or inhibiting the development of these cardiovascular diseases. 
Central in scavenging strong oxidants is the tripeptide glutathione (GSH) (10). 
Indeed, GSH depletion alone will result in development of severe hypertension 
(11). We have shown (12) that smooth muscle cells from spontaneously 
hypertensive rats (SHR), in comparison with those from normotensive rats, have 
increased oxidative stress that was correlated with lower GSH levels. We have 
also shown that increasing GSH content in SHR smooth muscle cells greatly 
decreased the oxidative stress in these cells (12). We decreased oxidative 
stress by exposing the cells to the phase 2 protein inducer sulforaphane. 
Sulforaphane (4-methylsulfinylbutyl isothiocyanate) is the most potent phase 2 
protein inducer found in our food sources (13). A major action of phase 2 
proteins, mainly enzymes, is to inactivate electrophiles and strong oxidants 
(14); hence, there has been a research interest in dietary phase 2 protein 
inducers as a means to prevent cancers (15). 
Previous SectionNext SectionMethods
We have argued that dietary phase 2 protein inducers should ameliorate 
conditions such as hypertension and atherosclerosis (16). The objective of the 
present research was to test this idea. The animal model chosen was the 
stroke-prone SHR (SHRsp). This strain develops severe hypertension (17) that is 
related to increased peroxynitrite formation (6) and develops inflammatory 
changes in blood vessels (18, 19). We used Sprague–Dawley (SD) rats as the 
normotensive controls. We chose broccoli sprouts as the source of phase 2 
protein inducer because certain cultivars of broccoli have been shown to be high 
in the glucosinolate (GS) glucoraphanin (Grn) that is metabolized into the phase 
2 protein-inducing isothiocyanate sulforaphane (13). GS are β-thioglucoside 
N-hydroxysulfates present in 16 families of higher plants with the distribution 
in the family Brassicaceae, particularly the genus Brassica, which is well 
described in ref. 20. Grn is not available in sufficient quantities to perform 
dietary experiments. We, therefore, chose to feed animals air-dried broccoli 
sprouts of the calabrese cultivar that we had determined were high in Grn and 
low in antinutritive GS. As a placebo control, we fed animals with broccoli 
sprouts that were frozen, thawed, and dried. This procedure allowed the 
myrosinase enzyme in the sprouts to access the GS and hydrolyze them (21). The 
basic difference between the two forms of dried sprouts is the presence of GS in 
the former and low levels of GS in the latter. 
Supporting Information. For additional details on methods, see Supporting 
Methods, which is published as supporting information on the PNAS web site. 
Broccoli Sprout Preparation and GS Measurements. Broccoli seeds, obtained from 
Mumm's Seeds and Sprouting (Shellbrook, Saskatoon, SK, Canada), were sprouted 
according to the supplier's instructions and screened for GS content by using 
HPLC, as outlined in ref. 22. For calibration purposes, a small amount of Grn 
was obtained from Ian McGregor (Agriculture and Agrifood Canada, Saskatoon 
Research Station, SK, Canada). The calabrese cultivar was selected because of 
its major Grn peak and low levels of antinutritive GS. We either air-dried 4-day 
broccoli sprouts at room temperature for 7 days before feeding the animals, or 
we froze, thawed, and dried them at 60°C. 
Phase 2 enzyme-inducing activity was measured by using a bioassay, as outlined 
in ref. 13, for the two broccoli sprout preparations. 
Animals and Tissue Preparation. We used male SHRsp (originally obtained from the 
National Institutes of Health, Bethesda, and maintained by the University of 
Saskatchewan Animal Facilities) and male SD rats (Charles River Breeding 
Laboratories, St. Constant, Quebec). All animals were treated in accordance with 
the Canadian Council on Animal Care Standards. We placed 5-week-old rats on 
AIN-93 defined diet (23), containing no synthetic antioxidants, and with an 18% 
casein-based protein content (Dyets, Bethlehem, PA). Each strain was divided 
into three groups (n = 8–10 rats per group) and treated for 14 weeks as follows: 
control diet (CT), AIN93 diet only; Grn+, AIN93 diet plus 200 mg of air-dried 
broccoli sprouts; Grn-, AIN93 diet plus 200 mg of broccoli sprouts in which GS 
were destroyed. Animals were housed individually, and 5 days each morning during 
the working week, the Grn+ and Grn- groups were fed 200 mg of the appropriate 
dried sprouts. Systolic blood pressures were measured biweekly by using an 
indirect tail-cuff plethysmography as described (24). 
At the end of study, anesthetized animals were perfused with buffered saline, 
and aorta, carotid artery, heart, and kidneys were removed. Some aorta tissues 
were used immediately for isometric tension studies, and the remaining tissues 
were frozen in liquid nitrogen. The frozen tissues were ground in the frozen 
state, and homogenized tissue powders were used for GSH analyses, enzyme assays, 
and Western blotting. 
Rats used for immunohistochemical studies were further perfused with a solution 
containing 4% paraformaldehyde. Aorta, carotid artery, heart, and kidney were 
dissected out, kept in same fixative overnight, frozen, cut into sections (10–12 
μm), collected on gelatin-chrome alum-coated slides, and stored at -20°C until 
further processing. 
Isometric Tension Studies. The thoracic aortae were cut into 2- to 3-mm rings 
and suspended under 1.0 g of tension in a 10-ml organ bath chamber filled with 
Krebs' saline solution aerated with 95%O2/5%CO2. Relaxation properties were 
measured as described (25). 
Measurements of GSH, Oxidized GSH (GSSG), and Enzyme Activities. GSH and 
oxidized GSH content were determined by using the HPLC method described in refs. 
26 and 27. The activities of GSH peroxidase (GPx), GSH reductase (GRed), and GST 
were measured as described (12). NQ01, NAD(P)H quinone reductase (QR) activity 
was measured as outlined in ref. 28. Protein content was determined by using the 
bicinchoninic acid procedure with BSA as reference (29). 
Immunohistochemistry. Frozen sections were immunostained by using primary Abs 
and either Cy3-fluorescence (Jackson ImmunoResearch) or the 
avidin–biotin–peroxidase (Vector Laboratories) detection systems. Primary Abs 
used were ED-1 (Serotec), marker for activated macrophages, and anti-NF-κB p65 
(Transduction Laboratories, Lexington, KY) that recognized the nuclear 
localization signal on p65. For quantification of inflammation in the kidney 
medulla, ED-1-positive cells were counted in 15 high-power microscopic fields 
(×400) per section, with six sections randomly selected per animal (n = 5 in 
each group). 
Western Blot Analysis. Proteins were separated on polyacrylamide gels by using a 
minivertical electrophoresis system (Bio-Rad) and transblotted onto 
polyvinyldifluoridene membranes, and membranes were then incubated with the 
appropriate primary Abs, followed by incubation with a horseradish 
peroxidase-conjugated secondary Ab (Bio-Rad) as done before (12). Proteins were 
visualized by using Chemiluminescent substrate kit (Amersham Biosciences) and 
quantified by using the un-scan-it gel automated digitizing system (version 5.1, 
Silk Scientific, Orem, UT). The membrane was reprobed with anti-β-actin (Sigma). 
Quantification was relative to the β-actin. For quantification of nitrosylated 
proteins, a mouse IgG mAb (Upstate Biotechnology, Lake Placid, NY) was used. For 
quantification of nuclearly localized NF-κB p65, nuclear extracts were obtained 
by using the NE-PER nuclear and cytoplasmic extraction kit (Pierce) as described 
(27). 
Data Analysis. Data are expressed as means ± SEM and analyzed by using Student's 
t test or ANOVA in conjunction with the Newman–Keuls test where applicable. 
Differences between groups were considered statistically significant at P < 
0.05. 
Previous SectionNext SectionResults
Broccoli Sprout Preparations. Our ability to deliver Grn, the GS precursor of 
the powerful phase 2 protein inducer sulforaphane, was limited by the 
unavailability of pure Grn in adequate quantities for chronic animal 
experiments. Young broccoli sprouts grown from suitable seeds are an extremely 
concentrated source of Grn (13). Slow air-drying of broccoli sprouts at room 
temperature for 7 days provides a dry product that is avidly consumed by rats, 
and was used for the Grn+ experiments. HPLC analyses of the 
desulfoglucosinolates obtained from extracts showed nine major GS peaks, of 
which seven were identified (see Fig. 7A, which is published as supporting 
information on the PNAS web site). Estimates of the relative amounts of GS by 
peak integration were as follows: glucoiberin, 3-(methylsulfinyl)propyl-GS, 
13.4%; Grn, 4-(methylsulfinyl)butyl-GS, 35.1%; glucobrassicanapin, 
4-pentenyl-GS, 8.0%; and gluconasturtiin, 2-phenethyl-GS, 14.1%. The 
antinutritive GS progoitrin (2-hydroxy-3-butenyl-GS, 2.8%) and sinigrin 
(2-propenyl-GS, 1.0%) were only minor components, as were the two unknowns 
(6.53% and 3.3%). By using desulfoglucoraphanin that we had collected by using 
HPLC as a reference, we estimated that these sprouts contained ≈12 μmol of Grn 
per gram of dry weight. In contrast, no GS were detected in extracts of broccoli 
sprouts that were frozen, thawed, and then dried at 60°C. The latter 
preparations were used for the Grn- diets (see Fig. 7B). 
To characterize these dried sprout preparations further, bioassays of the 
inducer potencies of extracts were carried out by described methods (13, 28). 
The Grn+ preparation treated with purified myrosinase contained 909,000 units of 
inducer activity per gram (equivalent to the inducer activity of 27.3 μmol of 
sulforaphane per gram). No inducer activity was detected in the absence of added 
myrosinase, indicating that little, if any, GS hydrolysis had occurred during 
the drying at room temperature. Because sulforaphane has ≈10 times greater 
inducer activity than other isothiocyanates (30) and Grn is the predominant GS 
in the preparation, one would have expected a better correlation between Grn 
quantification and sulforaphane-equivalents inducer activity. We now know that 
this discrepancy is due to suboptimal extraction of GS from our preparations 
during the analytical procedure (31). 
Extracts of the Grn- preparations, which contained no detectable GS by the 
desulfoglucosinolate HPLC method, had phase 2 protein inducer activities of 
16,700 and 76, 900 units per gram, before and after myrosinase treatment, 
respectively. These values correspond to the inducer equivalent of 0.5 and 2.31 
μmol sulforaphane, respectively. The Grn- preparation therefore contains ≈ 8.4% 
of the inducer activity of the Grn+ preparation, and there is, thus, a more than 
one order of magnitude difference in the GS content of these preparations. We 
conclude that the freezing, thawing, and drying at 60°C released the endogenous 
plant myrosinase, which hydrolyzed most of the GS to isothiocyanates, which were 
then inactivated probably by binding to proteins and other intracellular 
components. The Grn- preparation is, therefore, an appropriate control for the 
Grn+ preparation. 
Up-Regulation of GSH Levels and Down-Regulation of Oxidative Stress by Broccoli 
Sprout Consumption in SHRsp. In rats on CT, the basal levels of GSH in blood 
vessels, heart, and kidney were significantly lower in SHRsp than in SD (Fig. 1 
A and B). Although the Grn+ diet had no effect on GSH levels in SD tissues, it 
resulted in significant increases (≈1.5- to 2-fold) in SHRsp tissues (Fig. 1 A 
and B). The Grn- diet had no effect on GSH levels in the tissues of SHRsp. In 
contrast, GSSG levels were higher in these tissues in SHRsp than SD rats on CT 
(Fig. 1C). The Grn+ diet caused a significant decrease in tissue GSSG in SHRsp 
(Fig. 1C). The increase in GSH and decrease in GSSG caused by the Grn+ diet 
indicates a decrease in tissue oxidative stress. Congruent with increased 
oxidative stress was the observation that SHRsp kidney tissues had a prominent 
45-kDa nitrosylated protein band (see Fig. 8, which is published as supporting 
information on the PNAS web site). SHRsp on Grn+ diet had a 37% reduction in 
this nitrosylated protein compared with rats on control and Grn- diets. 
 
View larger version: 
  In this pageIn a new window
  Download as PowerPoint SlideFig. 1. Effect of Grn+ and Grn- diets compared with 
CT on GSH and GSSG in cardiovascular and kidney tissues of 19-week-old SHRsp and 
SD rats, n = 3–4 rats per group. Grn+ diet had no effect compared with CT in SD 
rats (A and B). SHRsp-CT and SHRsp-Grn- had significantly lower levels of GSH (A 
and B). SHRsp-CT had significantly higher levels of GSSG (C) in tissues examined 
compared with SD rats. When placed on the Grn+ diet, SHRsp rats demonstrated 
significant increases in GSH levels in all tissues examined (A and B). A Grn+ 
diet also decreased GSSG levels significantly in cardiovascular and kidney 
tissues in SHRsp (C). *, P < 0.05, for SHPsp controls or SHRsp-Grn- versus SD-CT 
and SD-Grn+; †, P < 0.05, for SHRsp-Grn+ versus SHRsp-Grn-. 
GSH-Related Enzyme and NQ01 NAD(P)H QR Activities. Basal activities of GPx (Fig. 
2A) and GRed (Fig. 2B) were significantly lower in aorta, carotid artery, heart, 
and kidney from SHRsp-CT than from SD-CT. In animals on the Grn+ diet, the 
enzyme activities of GPx and GRed were significantly higher than in SHRsp-CT 
(Fig. 2 A and B). There were no significant differences in GST in the 
cardiovascular or kidney tissues among the various experimental groups (data not 
presented). Because cardiovascular and kidney tissues were completely used in 
the other assays, QR activity was measured in liver. The activity was low in the 
male SHRsp, and the Grn+ diet did not affect the activity (14.6 ± 0.9 units in 
Grn+ and 14.5 ± 2.1 units in Grn-). In contrast, SD values were significantly 
higher in animals on Grn+ diet (96.6 ± 9.2 units) than on Grn- diet (60.8 ± 14 
units). 
 
View larger version: 
  In this pageIn a new window
  Download as PowerPoint SlideFig. 2. GPx and GRed activities in cardiovascular 
and kidney tissues of 19-week-old SHRsp and SD rats; n = 3–4 per group. GPx (A) 
and GRed (B) activities are significantly lower in all tissues examined in SHRsp 
on CT compared with SD rats on CT. SHRsp fed Grn+ diet had significantly 
increased GPx and GRed activities. *, P < 0.05, for SHRsp-CT versus SD-CT; †, P 
< 0.05, for SHPsp-Grn+ versus SHRsp-CT. 
Decrease of Macrophage Infiltration by Broccoli Sprout Consumption in SHRsp. In 
SHRsp-CT, activated macrophages could be identified in the inner intimal layers 
of the aorta, carotid artery, and endocardium of the heart, as well as in the 
kidney medullary interstitium and tubules (Fig. 3), indicating an inflammatory 
state in these structures. Few activated macrophages were seen in the 
cardiovascular system in SHRsp animals on Grn+ diet (Fig. 3 A–C). There were 
also significantly fewer (P = 0.026) activated macrophages in the kidney medulla 
of SHRsp on Grn+ diet (3.4 ± 0.9 cells per microscope field) than in SHRsp on 
Grn- diet (7.8 ± 1.0 cells per microscope field). Activated macrophages were not 
detectable in these tissues in the SD rats. 
 
View larger version: 
  In this pageIn a new window
  Download as PowerPoint SlideFig. 3. Representative micrographs demonstrating 
localization of activated macrophages (arrows indicate bright fluorescent cells) 
in the inner tissue linings of the aorta (A), carotid artery (B), and ventricle 
of the heart (C), as well as in the medulla of the kidney (D) in 19 week-old 
SHRsp on CT or Grn+ diet. Few such activated macrophages are present in tissues 
of animals on Grn+ diet. 
Decreased Activation of Nf-κB by Broccoli Sprout Consumption in SHRsp. 
Inflammation is associated with activation of the transcriptional factor complex 
NF-κB (32). In SHRsp on CT, nuclei containing NF-κB p65 were seen in the 
endocardium of heart (Fig. 4A) and tubules of the kidney medulla (Fig. 4B). Few 
cells expressed nuclearly localized NF-κB p65 in these organs in SHRsp on Grn+ 
diet. Western blot analysis demonstrated that nuclearly localized NF-κB p65 was 
significantly decreased by 40% in kidneys from SHRsp-Grn+ compared with 
SHRsp-CT. 
 
View larger version: 
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  Download as PowerPoint SlideFig. 4. Micrographs (A and B) and Western blot (C) 
showing the localization of activated NF-κB in SHRsp fed CT or Grn+ diet in 
heart ventricle (A) and kidney medulla (B and C) of 19-week-old animals. 
Positive nuclear NF-κB p65 signal was observed in ventricular endothelium and in 
the tubules of the kidney of SHRsp-CT with few positive cells seen in the 
SHRsp-Grn+ group. (C) Left shows a representative Western blot of nuclearly 
localized NF-κB p65 is shown on the left, whereas Right quantification of NF-κB 
p65 relative to β-actin (y axis depicts relative units). Data taken from kidney 
tissues of three animals per group. *, P < 0.05, for SHRsp-Grn+ versus SHRsp-CT. 

Prevention of Vascular Endothelium Dysfunction and Hypertension by Broccoli 
Sprout Consumption in SHRsp. Endothelial nitric oxide function was examined by 
precontracting aortic rings with phenylephrine, followed by exposure to 
acetylcholine (Fig. 5A). There was a concentration dependent decrease in tension 
in SD rats. There were no differences in relaxation with SD rats on CT, compared 
with Grn+ diet. In both cases there was an ≈0.9-g relaxation force. SHRsp-CT had 
a maximal relaxation of ≈0.33 g in response to acetylcholine, whereas rats on 
Grn+ diet had a significantly better mean relaxation of 0.70 g. SHRsp-Grn- had a 
mean relaxation of 0.57 g, which was significantly different from SHRsp-CT but 
only borderline significantly different (P = 0.0538) from SHRsp-Grn+. 
 
View larger version: 
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  Download as PowerPoint SlideFig. 5. Aortic-relaxation function in SD rats on CT; 
SD on Grn+ diet; and SHRsp on CT, Grn+ diet, and Grn- diet (n = 9 per group). 
Aortic rings were precontracted with phenylephrine, followed by administration 
of acetylcholine (ACh), and decrease in tension was measured. Diet had no effect 
on relaxation properties in SD rats. SHRsp-CT had significantly poorer 
relaxation responses to ACh than either of the SD groups. SHRsp-Grn+ had 
significantly better relaxation response to ACh than SHRsp-CT, whereas 
SHRsp-Grn- had an intermediate response. *, P < 0.001, for SHRsp-Grn- versus 
SD-CT and SD-Grn+. †, P < 0.001, for SHRsp-Grn+ versus SHRsp-CT. 
The ability of the nitric oxide donor sodium nitroprusside to cause a decrease 
in tension in aortic rings that were precontracted with phenylephrine was 
examined (see Fig. 9, which is published as supporting information on the PNAS 
web site). There was a dose-dependent decrease in tension in SD rats with a 
maximal decrease in tension of ≈0.98 g of force. The aortic rings obtained from 
all SHRsp diet groups had a significant impairment in relaxation in response to 
SNP with a maximal decrease in tension of ≈0.6 g. 
Systolic blood pressure was significantly lower in SD rats compared with SHRsp 
and did not change over the course of the experiment, regardless of diet. In 
SHRsp, the systolic blood pressure was 135 ± 4.6-mm Hg at the beginning of the 
dietary intervention (Fig. 6). This increased to 163 ± 1.8-mm Hg after 8 weeks 
on Grn+ and showed no further increase to the end of study. In contrast, blood 
pressures of SHRsp-CT and SHRsp-Grn- were significantly higher (184 ± 1.8-mm and 
182 ± 2.3-mm Hg, respectively) by the end of the study. 
 
View larger version: 
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  Download as PowerPoint SlideFig. 6. Systolic blood pressures over time in SD on 
CT; SD on Grn+ diet; and SHRsp on CT, Grn+ diet, and Grn- diet (n = 9 per 
group). No change in blood pressure is seen in SD over the 14-week period after 
weaning. Blood pressures progressively rise in all SHRsp groups for the first 8 
weeks on the postweaning diet and are significantly higher than in SD rats. From 
8 to 10 weeks, blood pressures rise further in SHRsp-CT and SHR-Grn- and then 
plateau. From 10 weeks of the dietary intervention onwards, blood pressure is 
significantly lower in SHRsp-Grn+ than in the two other SHRsp dietary groups. *, 
P < 0.05, for SD diet groups versus SHRsp diet groups; †, P < 0.05, for 
SHRsp-Grn+ vs. SHRsp-CT and SHRsp-Grn-. 
Previous SectionNext SectionDiscussion
SHRsp fed a Grn-containing diet had a major improvement in the cardiovascular 
system, as demonstrated by decrease in the rise in blood pressure and 
inflammation. Unlike the normotensive SD rats for which blood pressures remained 
constant during the experimental period, blood pressures in all three diet 
groups of SHRsp increased progressively until the animals were 13 weeks of age. 
Thereafter, animals on the Grn+ diet had significantly lower blood pressures 
than in the two other diet groups. 
The hypothesis initiating this research was that animals on a Grn+ diet would 
have decreased oxidative stress. This hypothesis was upheld. An index of 
oxidative stress is the GSH/GSSG ratio. Normotensive SD rats on control or Grn+ 
diets had ratios in the different tissues ranging from 17.1 to 20.9, SHRsp-CT 
had ratios ranging from 4.0 to 7.2, and SHRsp-Grn+ had significantly higher 
ratios ranging from 10.6 to 14.0. A decrease in oxidative stress with Grn+ is 
supported also by the increase in GPx activity in different tissues in 
SHRsp-Grn+. GPx is an enzyme that is susceptible to oxidative inactivation (33). 
Also in support of our hypothesis is the decrease in quantity of the 45-kDa 
nitrosylated protein band in kidney tissue. 
Higher oxidative stress in blood vessels should result in poorer endothelial 
function. Indeed, this effect is what we see with SHRsp on CT, as determined by 
ability of acetylcholine to cause only a 37% relaxation in 
phenylephrine-constricted aortae when compared with ability of SD aortae to 
relax. SHRsp on Grn+ diet had a response that was 78% of that of SD. 
Interestingly, SHRsp on Grn- diet had an endothelial cell response that was 
considerably better (68% of normotensive animals) than SHRsp on CT, but this 
better endothelial response did not translate into lower blood pressures. 
One source of oxidative stress is increased expression and activity of NAD(P)H 
oxidase that produces superoxide anion radicals (34). The expression of NAD(P)H 
oxidase proteins increases greatly after 15 weeks of age in both SHRsp and 
related SHR strain (35, 36). Inhibition of the activity of NAD(P)H oxidase has 
been shown to improve endothelial function (37). Hence, possibly what the Grn+ 
diet may be promoting is scavenging of such increased production of superoxide 
anion in the older SHRsp. It is known that endothelial nitric oxide synthase is 
also down-regulated in SHRsp and that inhibiting advanced glycation endproduct 
formation increases endothelial nitric oxide synthase expression (38). It is 
possible that the decreased vascular oxidative stress seen in our study also 
promoted endothelial nitric oxide synthase expression. The decrease of 11% in 
systolic blood pressure in SHRsp-Grn+ compared with SHRsp-CT is similar to the 
decrease in SHRsp blood pressure seen when oxidative stress was minimized by 
feeding rats a pharmacological inhibitor of advanced glycation endproduct 
formation (38). 
Higher oxidative stress should also increase activation of NF-κB promoting 
inflammatory responses. Indeed, we have shown that there is an increase in the 
activation of NF-κB in numerous tissues in SHRsp compared with SD rats and that 
a Grn+ diet decreases NF-κB activation. Decreased NF-κB activation correlated 
with decreased tissue infiltration of activated macrophages. Increased strong 
oxidant production in blood vessels and NF-κB activation plus infiltration of 
activated macrophages into tissues such as kidney is also seen in rats made 
hypertensive with deoxycorticosterone acetate (DOCA) (39). As with our study, 
DOCA rats exhibited infiltration of kidney tubules by activated macrophages. 
DOCA rats treated long-term with the synthetic antioxidants 
pyrrolidinedithiocarbamate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl have a 
decreased NF-κB activation and activated macrophage infiltration in kidneys 
(39). In these animals, the long-term administration of antioxidants also 
inhibited the rise in blood pressure. 
Are the effects seen in SHRsp fed Grn+ diet due to increased expression of phase 
2 proteins? Because of the centrality of GSH- and GSH-using enzymes in the 
antioxidant defense (10), we focused on this system. The rate-limiting enzyme 
for GSH synthesis, γ-glutamyl-cysteine ligase, belongs to the phase 2 gene 
synexpression group (40). That Grn+ diet had no effect on GSH levels in SD rats 
does not necessarily mean that γ-glutamyl-cysteine ligase was not induced 
because there is feedback inhibition of γ-glutamyl-cysteine ligase activity by 
GSH (41). That the increased GSH/GSSG ratio may be due to induction of phase 2 
proteins is supported by the observation that another phase 2 enzyme, GRed (42), 
is also up-regulated. However, QR activity was not induced in liver tissues in 
the SHR-Grn+ compared with SHRsp-CT, although we did demonstrate an increase in 
hepatic QR activity in the SD-Grn+ compared with SD-CT. Our findings are 
supportive of the idea that one consequence of the Grn+ diet is an up-regulation 
of phase 2 protein gene expression. 
Our results show that intake of broccoli sprouts high in Grn, whose metabolite 
sulforaphane is a potent phase 2 protein inducer, decreased oxidative stress and 
inflammation in kidneys and the cardiovascular system. The promotion of 
cardiovascular function is similar to that seen with long-term consumption of 
pharmacological antioxidants. In the present study, two doses were examined, 0.5 
and 5.5 μmol sulforaphane equivalents per rat per day. An effect on oxidative 
stress parameters and blood pressure was seen only with the higher dose, 
although some positive effects on endothelial function was seen with the lower 
dose. It may be true that even more profound effects might be seen with higher 
intake of Grn. Whether the positive effects seen are due to induction of phase 2 
proteins is not yet clear, and it may be true that Grn affects the GSH-related 
antioxidant system in some other way. 
In 1995, the direct costs of hypertension to the health care system in the 
United States of America was almost 20 billion dollars (43). Our results show 
that even a modest change in diet has the potential to have a major impact on 
health and reduce health care costs significantly. 
Previous SectionNext SectionAcknowledgments
We thank Arlene Drimmie, Anita Quon, Koleen Safiniuk, Connie Wong, and Yi Qian 
Wang for their excellent technical assistance. We especially thank Drs. J. W. 
Fahey and P. Talalay (The Johns Hopkins University School of Medicine, 
Baltimore) for their analyses of phase 2 enzyme-inducing activity in our sprout 
preparations, and Dr. Albena Dinkova-Kostova for advice on the QR assay. This 
work was supported by the Saskatchewan Agriculture Development Fund. 
Previous SectionNext SectionFootnotes
  ↵ ¶ To whom correspondence should be addressed at: Department of Anatomy and 
  Cell Biology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, 
  Canada S7N 5E5. E-mail: juurlink@duke.usask.ca. Abbreviations: CT, control 
  diet; GPx, GSH peroxidase; GRed, GSH reductase; Grn, glucoraphanin; GS, 
  glucosinolate; GSH, glutathione; GSSG, oxidized GSH; QR, quinone reductase; 
  SD, Sprague–Dawley; SHR, spontaneously hypertensive rats; SHRsp, stroke-prone 
  SHR. Copyright © 2004, The National Academy of Sciences
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  Published online before print April 21, 2004, doi: 10.1073/pnas.0402004101 
  PNAS May 4, 2004 vol. 101 no. 18 7094-7099 
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