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Sulforaphane improves the bronchoprotective response in asthmatics through Nrf2-mediated gene pathways


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Background: It is widely recognized that deep inspiration (DI), either before methacholine (MCh) challenge (Bronchoprotection, BP) or after MCh challenge (Bronchodilation, BD) protects against this challenge in healthy individuals, but not in asthmatics. Sulforaphane, a dietary antioxidant and antiinflammatory phytochemical derived from broccoli, may affect the pulmonary bronchoconstrictor responses to MCh and the responses to DI in asthmatic patients. Methods: Forty-five moderate asthmatics were administered sulforaphane (100 μmol daily for 14 days), BP, BD, lung volumes by body-plethsmography, and airway morphology by computed tomography (CT) were measured pre- and post sulforaphane consumption. Results: Sulforaphane ameliorated the bronchoconstrictor effects of MCh on FEV1 significantly (on average by 21 %; p = 0.01) in 60 % of these asthmatics. Interestingly, in 20 % of the asthmatics, sulforaphane aggravated the bronchoconstrictor effects of MCh and in a similar number was without effect, documenting the great heterogeneity of the responsiveness of these individuals to sulforaphane. Moreover, in individuals in whom the FEV1 response to MCh challenge decreased after sulforaphane administration, i.e., sulforaphane was protective, the activities of Nrf2-regulated antioxidant and anti-inflammatory genes decreased. In contrast, individuals in whom sulforaphane treatment enhanced the FEV1 response to MCh, had increased expression of the activities of these genes. High resolution CT scans disclosed that in asthmatics sulforaphane treatment resulted in a significant reduction in specific airway resistance and also increased small airway luminal area and airway trapping modestly but significantly. Conclusion: These findings suggest the potential value of blocking the bronchoconstrictor hyperresponsiveness in some types of asthmatics by phytochemicals such as sulforaphane.
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R E S E A R C H Open Access
Sulforaphane improves the bronchoprotective
response in asthmatics through Nrf2-mediated
gene pathways
Robert H. Brown
, Curt Reynolds
, Allison Brooker
, Paul Talalay
and Jed W. Fahey
Background: It is widely recognized that deep inspiration (DI), either before methacholine (MCh) challenge
(Bronchoprotection, BP) or after MCh challenge (Bronchodilation, BD) protects against this challenge in healthy
individuals, but not in asthmatics. Sulforaphane, a dietary antioxidant and antiinflammatory phytochemical derived from
broccoli, may affect the pulmonary bronchoconstrictor responses to MCh and the responses to DI in asthmatic patients.
Methods: Forty-five moderate asthmatics were administered sulforaphane (100 μmol daily for 14 days), BP, BD, lung
volumes by body-plethsmography, and airway morphology by computed tomography (CT) were measured pre- and
post sulforaphane consumption.
Results: Sulforaphane ameliorated the bronchoconstrictor effects of MCh on FEV
significantly (on average by 21 %;
p = 0.01) in 60 % of these asthmatics. Interestingly, in 20 % of the asthmatics, sulforaphane aggravated the
bronchoconstrictor effects of MCh and in a similar number was without effect, documenting the great heterogeneity
of the responsiveness of these individuals to sulforaphane. Moreover, in individuals in whom the FEV
response to MCh
challenge decreased after sulforaphane administration, i.e., sulforaphane was protective, the activities of Nrf2-regulated
antioxidant and anti-inflammatory genes decreased. In contrast, individuals in whom sulforaphane treatment enhanced
the FEV
response to MCh, had increased expression of the activities of these genes. High resolution CT scans disclosed
that in asthmatics sulforaphane treatment resulted in a significant reduction in specific airway resistance and also
increased small airway luminal area and airway trapping modestly but significantly.
Conclusion: These findings suggest the potential value of blocking the bronchoconstrictor hyperresponsiveness in
some types of asthmatics by phytochemicals such as sulforaphane.
Keywords: Asthma, Bronchodilation, Oxidative stress
Although corticosteroids are currently the mainstay of
asthma treatment, some asthmatics do not respond to
corticosteroids even at high doses [1] at which adverse ef-
fects impose therapeutic limitations. Moreover, corticoste-
roids are almost universally effective in controlling many
types of asthma, and this obscures the heterogeneity of
the disease [2]. Although inflammation may be an
important inciting factor, other molecular mechanisms of
airway disease may play a crucial role in asthma.
Antioxidant deficiencies have been associated with
poor asthma control and accelerated decline of lung
function [35]. Studies in mice suggest that disruption
of the nuclear factor erythroid-related factor 2 (Nrf2)
signaling pathway is a possible cause of chronic inflam-
mation, such as that associated with asthma [6]. Nrf2 is
responsible for maintaining and restoring cellular
homeostasis through antioxidant, anti-inflammatory, and
other mechanisms. Thus Nrf2 regulates critical enzymes
concerned with the biosynthesis of glutathione (GSH),
the dominant, small molecule, intracellular antioxidant.
A protective effect of GSH on inflammatory pathologies
* Correspondence:
Supported by: Flight Attendant Medical Research Institute (FAMRI)
Department of Anesthesiology and Critical Care Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD, USA
Division of Pulmonary Medicine and Critical Care, Department of Medicine,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
Full list of author information is available at the end of the article
© 2016 Brown et al.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (, which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain
Dedication waiver ( applies to the data made available in this
article, unless otherwise stated.
Brown et al. Respiratory Research (2015) 16:106
DOI 10.1186/s12931-015-0253-z
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
of the lung including asthma, has been demonstrated in
mice [7]. Another potentially critical role of the Nrf2 path-
way in asthma is highlighted by the dramatic increase in
eosinophilic airway inflammation, airway hyperreactivity,
and T helper cell 2 (Th2) cytokine production in Nrf2
knockout mice [6].
Sulforaphane, a phytochemical derived from cruciferous
vegetables, is a potent inducer of a variety of cytoprotective
(antioxidant and anti-inflammatory) enzymes (reviewed by
[8, 9]). It also inhibits the production of nitric oxide, coun-
teracts mitochondrial dysfunction and corrects low oxida-
tive phosphorylation, reduces lipid peroxidation, and
upregulates the heat shock response. In many tissues, sulfo-
raphane transcriptionally upregulates the genes encoding
cytoprotective enzymes primarily through the Keap1-Nrf2-
ARE system [9, 10]. Sulforaphane also potently inhibits in-
flammation by inhibiting the nuclear factor kappa-light-
chain-enhancer of activated B cells (NfκB) cascade [11], the
macrophage migration inhibitory factor (MIF), the p38
mitogen-activated protein kinase (MAPK) activation cas-
cade, and by modulating β-catenin signaling [1214].
Unlike direct antioxidants such as vitamin C, sulforaph-
ane is an indirect antioxidant and upregulates multiple
antioxidant pathways without being consumed in the anti-
oxidation process [15]. Sulforaphane boosts the antioxidant
capacity of cells by at least two major indirect mechanisms:
by induction of phase 2 cytoprotective enzymes and by
dramatically increasing cellular GSH levels. Antioxidant
deficiencies have been strongly associated with poor
asthma control and accelerated lung function decline, and
represent a reasonable therapeutic target [1618].
Nrf2-mediated signaling pathways limit airway eosino-
philia, mucus hypersecretion, and airway hyperresponsive-
ness to allergen challenge in a murine model of asthma
[6]. Genetic disruption of the Nrf2 gene (knock-out
models) leads to severe allergen-driven airway inflamma-
tion and hyper-responsiveness in mice [6]. An et al. [19]
recently showed that airway smooth muscle cells isolated
from Nrf2
mice exhibited significantly higher contract-
ile force compared to cells isolated from Nrf2
Thus, there is strong evidence for the role of oxidative
stress, mediated through dysfunction of the Nrf2 pathway,
as a mechanism of airway hyper-responsiveness character-
istic of asthma.
Sulforaphane can be safely and consistently administered
to humans by feeding a broccoli sprout extract in which its
precursor glucosinolate (glucoraphanin) has been enzymati-
cally hydrolyzed [9]. Sulforaphane has very low toxicity. Its
administration is well tolerated and it is extensively con-
sumed as a component of cruciferous vegetables. It may be
considered a food, a dietary supplement, or a drug, depend-
ing on dosage and its intended use [9, 20, 21].
It has been known for more than 20 years that deep
inspiration (DI) protects against MCh induced bronchial
constriction. This protective response is characteristic-
ally absent in even mild asthmatic patients [22, 23], and
indeed, DI can even exacerbate the bronchoconstrictor
effects of MCh in asthmatics [24]. The beneficial effects
of DIs occurring before or after MCh challenge have
been designated bronchoprotection (BP) and broncho-
dilation (BD), respectively [22, 25]. Elucidation of the
pathways leading to the loss of the BP and BD responses
in asthma may give further insight into the disease
process and reveal new treatments.
The current study was designed with the primary ob-
jective to test the hypothesis that sulforaphane could
augment the deep inspiration (DI)-induced bronchopro-
tection (BP) and bronchodilation (BD) responses in indi-
viduals with airways hyperresponsiveness. Secondary
objectives included testing the hypotheses that sulfo-
raphane would also affect pulmonary function and lung
morphology as shown by high resolution CT scans.
The protocol was approved by the Johns Hopkins
IRB and written informed consent was obtained
(NA_00011275) and registered at
(NCT00994604). A total of 51 individuals with airway
hyperresponsiveness were screened who reported
upper or lower respiratory symptoms (or both) in the
absence of upper respiratory infections in the previous
12 months. Five subjects withdrew, and one subject
failed the screen. Subjects were also required to have
70 % of predicted values, and a positive con-
ventional multi-dose methacholine (MCh) inhalation
challenge (PC
provocative concentration causing a
20 % drop in FEV
)of25 mg/ml, as well as a BP ef-
fect of DI of less than 40 %. Short-acting or long-
acting bronchodilators were withheld before MCh
challenge for 12 or 48 hours, respectively. The demo-
graphic and baseline pulmonary functions of smokers
(n = 15) and non-smokers (n = 30) were similar (Table 1).
Thirty-one subjects were taking albuterol, 13 were tak-
ing fluticasone/salmeterol, 8 were taking fluticasone
alone, 3 were taking montelukast, 1 was taking beclo-
methasone, 1 was taking budesonide/formoterol, 3
were taking fluticasone nasal spray, 4 were taking lora-
tadine, 1 was taking fexofenadine, 1 was taking cetiri-
zine, and 1 was taking benedryl. All participants were
at least 45 days past the end of their most recent URI
for all of their visits, and were asked to withhold inhaled
bronchodilators prior to each study visit: 12 hours for
short-acting and 48 hours for long-acting beta agonists.
Full analysis was possible in 44 individuals. Exclusion cri-
teria included persistent respiratory symptoms suggesting
uncontrolled asthma.
Brown et al. Respiratory Research (2015) 16:106 Page 2 of 12
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Pulmonary function measurements
All subjects underwent baseline spirometry, body-
plethysmography, diffusing capacity of the lung for carbon
monoxide, and exhaled nitric oxide (eNO) measurements.
For spirometry, we used Hankinson's (NHANES III) pre-
dicted values [26]. We further made the widely accepted
assumption that the reproducibility of FEV
values was
0.1 L [27, 28], so that x-axis changes in Fig. 2a and b be-
tween 0.1 and +0.1 L were considered as no effect. In
addition, blood was obtained before and immediately after
sulforaphane administration to monitor standard blood
chemistries for safety (n = 45) and to measures mRNA ex-
pression of Nrf2 target genes in peripheral blood mono-
nuclear cells (n = 35).
Evaluation of beneficial effects of deep inspiration (DI)
DI-induced bronchodilation was performed by using
multiple modified single dose MCh challenges on separ-
ate days [22, 25, 29]. Briefly, after baseline spirometry, at
every single dose challenge, study participants were
instructed to abstain from DI for 20 min. At the end of
this period, a single dose MCh challenge (starting at
0.025 mg/ml) was delivered with five tidal inspirations
from a deVilbiss 646 nebulizer attached to a model 2A
Rosenthal-French dosimeter (Laboratory for Applied Im-
munology, Fairfax, VA). Three minutes later, a single full
spirometric maneuver was performed and the degree of
airways obstruction was calculated by comparing base-
line to post MCh FEV
. If the MCh-induced reduction
in FEV
was less than 20 %, the participant was asked to
return on a separate day for another single dose MCh
challenge, using the next highest single dose of MCh
(e.g. 0.075 mg/ml). This process was continued with
additional single dose challenges (0.25, 0.75, 2.5,
7.5 mg/ml) on separate days, until the single dose in-
ducing 20 % reduction in FEV
was achieved. For the
challenge at this level, where 20 % or greater reduction
in FEV
was obtained, the participant was instructed
to continue the procedure by taking 4 DI immediately
after the single post-MCh spirometry. Another spiro-
metric maneuver was performed immediately after the
4 DI to calculate the degree to which the participant
was able to reverse the MCh-induced airway obstruc-
tion (Figure below).
Measuring the difference between the post-MCh FEV
and the FEV
obtained after the 4 DI, we calculated a
measure of bronchodilation induced by the DI, which
we termed the bronchodilation (BD) index. This meas-
ure is calculated as follows:
BD index ¼ð1ðð1ððFEV1af ter MCh and af ter DIÞ
ðFEV1baselineÞÞÞ  ð1ððFEV1af ter MChÞ
ðFEV1baselineÞÞÞÞÞ  100
Essentially, the BD index is derived from two compo-
nents, the reduction in FEV
from baseline after MCh
and after DI and the reduction in FEV
from baseline
after MCh, but before DI.
On a separate day, the same single MCh dose used to
achieve a 20 % or greater reduction in FEV
was again
Table 1 Baseline demographics and pulmonary functions (mean ± SD) of 45 asthmatic patients
Non-smoker Smoker All
Number of subjects (n) 30 15 45
Male/female 10/20 9/6 19/26
Age (mean ± SD) 38 ± 14 40 ± 13 38 ± 13
Smoking (pack-years) 0 11 ± 10 -
Race (black/white/asian) 20/9/1 10/5/0 30/14/1
(provocative concentration causing a 20 % drop in FEV
, in mg/ml) 2.4 ± 4.2 2.6 ± 2.1 2.4 ± 3.6
(forced expiratory volume in 1-second, in L) 2.7 ± 0.7 2.8 ± 0.8 2.7 ± 0.7
% predicted (forced expiratory volume in 1-second) 87 ± 10 % 91 ± 13 % 88 ± 11 %
FVC % predicted (forced vital capacity) 90 ± 12 % 97 ± 15 % 92 ± 13 %
/FVC 0.80 ± 0.08 0.78 ± 0.01 0.79 ± 0.08
TLC (total lung capacity, in L) 5.0 ± 1.4 5.1 ± 1.2 5.0 ± 1.4
SVC (slow vital capacity, in L) 3.3 ± 1 3.5 ± 0.9 3.4 ± 1.0
FRC (functional residual capacity, in L) 3.0 ± 0.9 3.3 ± 1.5 3.1 ± 1.1
RV (residual volume, in L) 1.7 ± 0.7 1.7 ± 0.8 1.7 ± 0.7
RV/TLC 0.34 ± 0.09 0.33 ± 0.08 0.34 ± 0.09
Diffusing capacity of carbon monoxide (mL/min/mm Hg) 22.5 ± 5.9 22.9 ± 5.4 22.7 ± 5.6
Specific airway resistance, (in kiloPascals · s) 6.1 ± 3.4 6.0 ± 3.4 6.04 ± 3.3
Exhaled nitric oxide (ppb) 10.4 ± 11 6.15 9.1 ± 10
Brown et al. Respiratory Research (2015) 16:106 Page 3 of 12
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administered after 20 min of quiet breathing, followed by 5
DI to prevent MCh-induced obstruction (Figure below).
Assessing the difference between the MCh-induced re-
duction in FEV
on the day no DI were taken to the day
on which 5 DI were taken prior to the challenge, we ob-
tained a measure of bronchoprotection induced by the
series of DI, which we termed the bronchoprotection
(BP) index which has been previously described [22, 29].
This measure is calculated as follows:
BP index ¼ð1ðð1ððFEV1af ter DI s and af ter MChBÞ
ðFEV1baselineBÞÞÞ  ð1ððFEV1af t er MChAÞ
ðFEV1baselineAÞÞÞÞÞ  100
The BP index is derived from two components, the
MCh-induced reduction in FEV
from baseline on the day
that 5 DI preceded the single-dose MCh challenge and the
MCh-induced reduction in FEV
from baseline on the day
no DI were taken before the single-dose MCh challenge.
Acquisition and analysis of CT scans
All scans were performed with a single spiral CT scanner
(Siemens, Definition 64) with settings of 120 Kilovolt peak.
mAs (milliampere · second) was based on body size (small
= 80 mAs, medium = 100 mAs, large = 145 mAs), with a ro-
tation time of 0.5 s, pitch of 1.0 mm, thickness of 0.75 mm,
and interval of 0.5 mm. Images were reconstructed using a
B35 and B31 algorithm. All subjects were coached and
practiced the breathing maneuvers before scanning. For the
total lung capacity scans, while in the scanner, all subjects
were instructed to take a deep breath and blow it out. This
maneuver was repeated three times. On the third deep in-
spiration, the subjects were instructed to hold their breath.
They were then coached to continue to keep holding their
breath for the <10 s duration of the scan. For the functional
residual capacity (FRC) scans, all subjects were similarly
instructed to take a breath in and blow it out. This maneu-
ver was repeated three times. On the third time, the subject
was instructed to expel their breath and then hold their
breath for the duration of the scan (<10 s). Lung volumes
and airway dimensions were calculated using PW software
(VIDA Diagnostics, Inc. Coralville, IA) based on the lung
CT scans. The PW software calculates the total lung volume,
the lung air volume, the lung tissue volume, the lung density
in Hounsfield Units (HU), air trapping (% voxels < 856
HU), the luminal diameter, the wall thickness and the wall
fraction (wall area/total airway area). In addition, the airways
were arbitrarily divided into three groups (small, medium,
and large) of a similar number of airways per group.
Preparation and administration of sulforaphane-rich
broccoli sprout extract
The sulforaphane-rich broccoli sprout extract was prepared
at the Lewis B. and Dorothy Cullman Chemoprotection
Brown et al. Respiratory Research (2015) 16:106 Page 4 of 12
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Center at Johns Hopkins University, essentially as previ-
ously described [30, 31]. We obtained IND approval from
the FDA for its use in this study (IND #79274). In brief,
specially selected BroccoSproutsseeds were surface-
disinfected and grown for 3 days in a commercial sprouting
facility under controlled light and moisture. A boiling water
extract was prepared, filtered, cooled, and treated with the
enzyme myrosinase (from daikon sprouts) in order to con-
vert precursor glucosinolates to isothiocyanates, and then
lyophilized at a food processing facility (Oregon Freeze Dry,
Albany, OR). The lyophilized powder (227 μmol sulforaph-
ane/g powder) was packaged in sterile plastic tubes that
were unitized for daily doses by ALFA Specialty Pharmacy
(Columbia, MD); each tube contained 100 μmol sulforaph-
ane (440 mg of BSE). The powders (bulk and individual
doses) were maintained at 20 °C, repeatedly checked for
microbial contaminants and sulforaphane titer before con-
veyance to the study site to be dispensed to patients.
Study subjects were given both verbal and written in-
structions to avoid eating any food products that contain
the following vegetables (either cooked or raw) until com-
pletion of the study: Cruciferous vegetables (such as broc-
coli, broccolini, broccoli raab, rapini, kale, cabbage, brussels
sprouts, cauliflower, arugula, turnips, radish, turnip, turnip
greens, kohlrabi, rutabaga, mustard greens, collard greens,
chinese cabbage, pak choi, bok choi, napa, watercress, broc-
coli sprouts, daikon, sauerkraut, coleslaw), vegetables in the
onion family (onions, leeks, garlic, or chives), and the fol-
lowing condiments: mustard, horseradish, wasabi, soy
sauce, or Worcestershire sauce.
mRNA gene expression
Nrf2regulated antioxidative genes were measured in per-
ipheral blood mononuclear cells with quantitative real-time
PCR (qRT-PCR). Total RNA was extracted from the cells
by use of the Qiagen RNeasy kit (Qiagen, Valencia, CA).
Total RNA was used for cDNA synthesis with random hex-
amers and MultiScribe reverse transcriptase, according to
the manufacturers recommendations (Applied Biosystems).
cDNA (100 ng) was used for quantitative PCR analyses of
selected genes: glutamate cysteine ligase catalytic (GCLC)
and modifier subunits (GCLM), NAD()H-quinone oxidore-
ductase 1 (NQO1), and glutathione S-transferase-1 (GST1)
by using primers and probe sets commercially available
from Applied Biosystems. Assays were performed by using
the ABI 7000 Taqman system (Applied Biosystems). β-
Actin was used for normalization.
After the baseline assessment of BP and BD, spirom-
etry, body-plethysmography, diffusion capacity of carbon
monoxide, exhaled NO, and CT scan measurements
were completed (Fig. 1), volunteers received a 1-week
supply of 20-ml bottles containing 100 μmol of sulfo-
raphane each dissolved in mango juice, which masks the
pungency of the BSE [20], to be stored in a freezer. Each
morning, a single bottle was allowed to thaw at room
temperature until consumption of the contents in the
evening. The subject then received a second 7-day sup-
ply from the laboratory, and the procedure was repeated.
At the end of this period, BP, BD, CT scan, spirometry,
body plethysmography, diffusion capacity of carbon
monoxide, and exhaled NO were re-assessed within
3 days of consumption of the last bottle (Fig. 1). In total,
the subjects consumed 100 μmol of sulforaphane per
day on 14 consecutive evenings.
Safety and adverse event monitoring
Adverse event monitoring and documentation by severity,
duration, and relatedness were performed by the study
Fig. 1 Time line of trial protocol
Brown et al. Respiratory Research (2015) 16:106 Page 5 of 12
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physician at each visit. Standard laboratory chemistries
were drawn at baseline and after the two weeks of con-
suming sulforaphane. Study stopping rules were based on
the standard practice of our local IRB and the FDA. Study
drug safety and adherence to the protocol were monitored
annually by the IRB.
Data analysis was performed with JMP 11.0.0 software
(SAS Institute, Cary, NC). To compare the demographic
and baseline pulmonary function data, one-way ANOVA
and Chi-squared tests were used. To compare the effects
of sulforaphane on changes in BP and BD, lung function,
CT measurements, oxidative stress genes, and effects of
smoking, matched-pairs comparisons (pre- and post-
sulforaphane) with non-parametric Wilcoxon Sign Rank
Analyses were performed. In addition, one-way ANOVA,
and simple linear regressions were used where indicated.
To examine the effects of specific genes and changes in
interaction on the BP, we constructed a multivariate
regression model. Significance was assumed at p 0.05.
The time-line of the protocol for this study is presented
in Fig. 1.
Screening and selection of asthmatic subjects
A total of 51 individuals with airway hyperresponsive-
ness were screened. The demographic and baseline pul-
monary functions as shown in Table 1. Full analysis was
possible in 44 individuals.
Primary outcomes
The primary goal of these studies was to determine
whether sulforaphane administration to 44 asthmatic pa-
tients: (i) affected the magnitude of the bronchoconstric-
tor effect of single doses of MCh challenge, and (ii)
affected the protective effects of deep inspiration on the
magnitude of the bronchoconstrictor effect of MCh
challenge. The final metric in all these studies was FEV
The mean reduction in FEV
resulting from a single
dose MCh challenge was 28.7 ± 7.2 % (mean ± SD)
(Table 2), but individual asthmatics varied markedly in
their responses to sulforaphane (Fig. 2a and b). Whereas
in 60 % of the asthmatics sulforaphane blocked the
bronchoconstrictor effects of MCh challenge, in 20 % of
these subjects sulforaphane aggravated the bronchocon-
strictor effect of MCh, and in a similar proportion (20 %)
it had no effect.
When the change in FEV
response to MCh was
analyzed post hoc as either a decrease or an increase
in response to sulforaphane administration, the result-
ing BP and BD responses were dramatically different.
Those subjects whose FEV
response to MCh de-
creased (13 ± 8 %), had a concomitant reduction in
BP response (p = 0.002; Table 3), whereas those sub-
jects whose FEV
response to MCh increased (11 ±
11 %), experienced an improvement in BP (p = 0.004,
Table 3). Changes in BD paralleled these changes in
BP, (p = 0.02 for the subjects whose FEV
response to
MCh decreased and p = 0.04 for the subjects whose
response to MCh increase; Table 3). The impli-
cation of these paradoxical responses is not yet clear
and the mechanism is not understood.
Secondary outcomes
Secondary outcomes examined changes in pulmonary
function (Table 4) and lung morphology as determined
by high-resolution CT scans (Table 5). We found a sig-
nificant reduction in specific airway resistance (p = 0.03,
Table 4), and a small but significant increase in the small
and medium airway luminal area (p = 0.03, Table 5).
Gene-dose effects
Peripheral blood mononuclear cells were obtained from
35 subjects for the measurement of Nrf2regulated anti-
oxidant genes, and we were able to compare 28 sample
Table 2 Effects of sulforaphane (SF) administration and deep inspiration on A. Changes in forced expiratory volume in 1-second
) produced by the bronchoconstrictor MCh, and B. Magnitudes of bronchoprotection (BP) and bronchodilation (BD) effects
A. Treatment % Change in FEV
(mean ± SD)
Before SF After SF p for SF effect
MCh 28.7 ± 7.2 22.7 ± 12.7 0.006
MCh preceded by 4 deep inspirations (for BP)23.0 ± 13.4 19.1 ± 11.9 0.04
MCh followed by 4 deep inspirations (for BD)18.4 ± 7.7 16.3 ± 10.7 0.34
B. Magnitudes of deep inspiration effects Change in BP and BD (mean ± SD)
Before SF After SF p for SF effect
BP 15.5 ± 52.7 10.3 ± 57.1 0.83
BD 32.5 ± 32.7 26.7 ± 30.6
One value was outside the acceptable range (>2 S.D.) and has been censored
Brown et al. Respiratory Research (2015) 16:106 Page 6 of 12
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pairs before and after sulforaphane treatment. When
gene activities were classified according to the changes
in FEV
(either a decrease or an increase response to
MCh after sulforaphane administration), the results were
very different. When the FEV
response to MCh
decreased after sulforaphane administration, the activ-
ities of all three Nrf2-regulated genes (NQO1, HO1,
GCLM) decreased. In contrast, when the FEV
to MCh increased after sulforaphane administration, the
gene activities of all three genes increased (Table 6).
Comparing the changes in each gene expression with
changes in FEV
response to sulforaphane treatment, the
differences in gene expression was significant for GCLM
(p = 0.03) and NQO1 (p = 0.047).
Bronchoprotection (BP), bronchodilation (BD), and gene
The BP and BD responses were also analyzed according
to changes in gene expression (either a decrease or an
increase resulting from sulforaphane administration).
There was a significant relationship between the change
in NQO1 gene expression and BP (p = 0.03). When
NQO1 decreased after sulforaphane administration, the
BP worsened (54.4 ± 78.3 %) and when NQO1 in-
creased after sulforaphane administration, the BP im-
proved (20.6 ± 72.8 %). There were no statistically
significant relationships for the other genes or for BD.
We next examined the potential interactions among
these factors. A multivariate regression model was con-
structed using the change in BP as the outcome variable.
Independent variables were the changes in MCh-
induced decrease in FEV
(the difference between base-
line before and after sulforaphane treatment), and the
change in GCLM, GST1, and NQO1 gene expression.
The overall model was significant (r
= 0.67, p = 0.0003).
Controlling for the other variables, there was a signifi-
cant negative correlation between the change in BP and
the change in FEV
from baseline (p = 0.002), and a sig-
nificant positive correlation between the change in BP
and the change in NQO1 gene expression (p = 0.007).
Bronchoprotection, bronchodilation, and smoking history
When BD and BP responses in the non-smokers and
smokers were partitioned by the change in FEV
sponse to MCh, they mirrored the overall changes. In
those subjects whose FEV
response to MCh de-
creased whether they were non-smokers or smokers,
there was a concomitant reduction in BP and BD re-
sponses (Table 7). Conversely, subjects whose FEV
sponse to MCh increased whether they were non-
smokers or smokers, showed improvement in BP and
BD values (Table 7).
Safety and tolerance
Sulforaphane treatment was safe and well-tolerated. In-
spection of the laboratory results among all the subjects
showed that only one set of laboratory values in one
subject showed a significant liver function (transamin-
ase) elevation after sulforaphane administration. On
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
Effects of SF on the change in FEV1 (L) caused by MCh
Effects of SF on BD (% change)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
Effects of SF on the change in FEV1 (L) caused by MCh
Effects of SF on BP (% change)
Fig. 2 Negative correlation between the changes (%) resulting from
sulforaphane (SF) administration on: a. bronchodilation (BD) and on
b. bronchoprotection (BP) in asthmatic subjects, and the effects of
sulforaphane administration on the reduction of FEV
caused by
MCh challenge. a: There was a significant negative correlation
between the changes in BD and the changes in FEV
= 0.13,
p = 0.01). As the decrease in FEV
with MCh challenge (airway
narrowing) became larger with administration of sulforaphane, the
BD response became smaller. b: There was a significant negative
correlation between the changes in BP and the changes in FEV
= 0.26, p = 0.0005). As the decrease in FEV
with MCh challenge
(airway narrowing) became larger with administration of
sulforaphane, the BP response became smaller. The Mean control
was 2.7 ± 0.7 L, and a single MCh challenge caused a reduction
in FEV
by 0.78 ± 0.3 L. We further made the widely accepted
assumption that the reproducibility of FEV
values was 0.1 L, so that
x-axis changes in Fig. 2a and b between 0.1 and +0.1 L were
considered as no effect
Brown et al. Respiratory Research (2015) 16:106 Page 7 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
further questioning, the subject reported that he had run
a marathon the day before the blood draw after sulfo-
raphane treatment, and this may have been responsible
for changes in liver enzymes. On repeating the blood
chemistries, one week later, all blood chemistries were
within normal limits.
To our knowledge, this is the first study that has ex-
amined the effects of the phytochemical sulforaphane
on the response to DI in individuals with asthma.
This study addressed the hypothesis that oxidative
stress and inflammation play roles in the mechanisms
of the beneficial effects of DI. While our a priori pri-
mary outcome was negative, post hoc analysis exam-
ining the groups according to whether they had an
increase or a decrease in FEV
response to Mch told
a very different story.
When sulforaphane attenuated the decline in FEV
lowing a single MCh challenge, BP worsened. In con-
trast, if the FEV
response to a single MCh challenge
remained the same or increased slightly after sulforaph-
ane treatment, BP improved dramatically. There were
similar changes for the BD responses. In addition when
sulforaphane increased NQO1 gene expression, there
was an associated increase in FEV
response to MCh
and independently associated improvement in the BP re-
sponse of DI in this cohort of asthmatics.
It is predictable that there should be a range of sulfo-
raphane responses among individuals, and that they be
related to some other biological phenomenon that we
have yet to understand. For example, we have observed
striking demonstrations of this effect in our clinical
studies of sulforaphane effects on autism spectrum dis-
order behavioral metrics in young men [31], and in clin-
ical evaluation of the effect of sulforaphane on both
aflatoxin and air pollutant metabolites [30, 32].
It is not yet fully explainable (although expected), that
multiple mechanisms could be at play in creating this
range of responses. The literature suggesting a bi-phasic
response, commonly referred to as the U-shaped or J-
shaped curve, or hormesis [33, 34] and well described in
the context of phytochemicals [35], points to many such
examples in which a compound may have a protective
or therapeutic effect at one concentration and a toxic or
detrimental effect at another higher level of exposure.
This is well demonstrated in the case of Nrf2-active
compounds, in particular in their effects on redox sig-
naling, and differential pro- and anti-oxidant activities
that are concentration dependent [36].
A limited number of animal and human studies have
examined the effects of sulforaphane on airway respon-
siveness. Park and colleagues showed that sulforaphane
Table 3 The BP and BD responses to sulforaphane treatment, classified according to initial increased or decreased forced expiratory
volume in 1-second (FEV
) response to MCh (mean ± SD). Wilcoxon sign rank test
Before SF After SF p-for SF effect Before SF After SF p-for SF effect
Overall FEV
(from Table 2) 15.5 ± 52.7 10.3 ± 57 0.82 32.5 ± 32.7 28.2 ± 28 0.33
Decreased FEV
response to MCh
(n = 29)
37.6 ± 38.0 0.8 ± 64 0.002 38.3 ± 34.6 23.2 ± 35.1 0.02
Increased FEV
response to MCh
(n = 15)
27.2 ± 51.7 31.7 ± 33 0.004 19.6 ± 25.1 33.4 ± 18.6 0.04
Table 4 Change in pulmonary function with sulforaphane (SF) treatment (mean ± SD)
Before SF After SF p for SF effect
% predicted (forced expiratory volume in 1-second) 88 ± 11 % 88 ± 11 % 0.79
FVC % predicted (forced vital capacity) 92 ± 13 % 92 ± 11 % 0.95
/FVC 0.79 ± 0.08 0.77 ± 0.01 0.56
TLC (total lung capacity, in L) 5.0 ± 1.4 4.9 ± 13 0.10
SVC (slow vital capacity, in L) 3.4 ± 1.0 3.3 ± 0.9 0.38
FRC (functional residual capacity, in L) 3.1 ± 1.1 3.1 ± 1.0 0.56
RV (residual volume, in L) 1.7 ± 0.7 1.6 ± 0.7 0.56
RV/TLC 0.34 ± 0.09 0.34 ± 0.1 0.97
Diffusing capacity of carbon monoxide, (mL/min/mm Hg) 22.7 ± 5.6 22.4 ± 5.7 0.46
Specific airway resistance (kiloPascals · s) 6.04 ± 3.3 4.8 ± 2.5 0.008
Exhaled nitric oxide (ppb) 8.8 ± 10 9.3 ± 10.9 0.80
Brown et al. Respiratory Research (2015) 16:106 Page 8 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
inhibits ovalbumin-induced airway inflammation in a
murine model of asthma [37]. They further surmised
that this occurred though suppression of GATA-3 and
SOCS-3 and increased T-bet and SOCS-5 expression.
Whether this also occurs in humans has not been deter-
mined. Nrf2antioxidant response element binding was
reduced in cultured airway smooth muscle cells from pa-
tients with severe asthma compared to similar cells from
patients with moderate asthma or with those from nor-
mal subjects [38]. How this affects the asthmatic re-
sponse in vivo is unknown.
Sulforaphane effects on upper airway inflammation,
and on airway response to challenge, have also been
measured. Oral sulforaphane induced airway Phase 2
antioxidant enzyme expression in a dose-dependent
fashion in exfoliated human upper airway cells [39]. In a
follow-up study, when sulforaphane in the form of a
broccoli sprout extract was administered orally for 4 days
before a diesel exhaust particle challenge, total cell count
in the nasal lavage was reduced by more than 50 % [40].
These studies establish that the oral administration of
sulforaphane is an effective means of inducing cytopro-
tective enzymes in the upper airway and by inference,
probably also in the lower airway. In addition, the doses
in these published studies were similar to those used in
the current study and thus support our choice of dosing
regimen. We chose to administer the sulforaphane for a
longer period of time, 14 days rather than 4 days, in
order to be confident that enough time had elapsed for
substantial enzyme induction.
It is well recognized that pro-inflammatory cytokines,
chemokines, as well as other cell mediators, play import-
ant roles in the allergic inflammatory process in asthma.
Dysfunction of the Nrf2 pathway has also been linked to
severe allergen-driven airway inflammation and hyper-
responsiveness in mice [6]. Disruption of the Nrf2 sig-
naling pathway in mouse models has also been directly
linked to chronic inflammation such as that associated
with asthma [6]. As a potent inducer of cytoprotective
enzymes (antioxidant and anti-inflammatory) via Nrf2,
sulforaphane thus directly affects both the antioxidant
deficiencies that have been associated with poor asthma
control and accelerated lung function decline [1618],
and the inflammatory component of these conditions
[1214], both of which represent attractive therapeutic
Upregulation of Phase 2 enzymes and of other Nrf2
targets by sulforaphane has previously been shown in
vitro and in animal models [9, 39, 4143]. The use of
broccoli sprout extract as a vehicle for well-quantified
oral delivery of sulforaphane permitted us to examine
the role of oxidative stress mediated through the Nrf2-
signaling pathways on lung function in asthma. Edible
plants contain a wide variety of phytochemicals, some of
which are phase 2 enzyme inducers. Large quantities of
inducers of enzymes that are potent antioxidants can be
delivered in the diet by small quantities of broccoli
sprouts (e.g., three-day-old sprouts) that contain as
much inducer activity as 10100 times larger quantities
of mature broccoli. A well-characterized, stable, clean,
standardized, homogeneous, sulforaphane-rich food ex-
tract can be produced and utilized across a range of cell
culture, animal, and clinical studies, permitting direct
comparison of results. Thus, a large body of evidence
has accumulated around the world in a variety of experi-
mental systems suggesting that sulforaphane is the active
agent in broccoli sprout extracts responsible for almost
all of the phase 2 response that is induced following
treatment with broccoli sprout extracts. Quantifiable
and reproducible extraction of sulforaphane from BSE
has thus permitted us to target specifically the Nrf2
pathway in order to elucidate the mechanisms of oxida-
tive stress in patients with asthma.
Table 5 CT scan measurements of changes in volume, luminal
area and airway wall thickness with sulforaphane (SF) treatment
measured at either total lung capacity (TLC) or functional
residual capacity (FRC) (mean ± SD)
Before SF After SF p for SF effect
TLC lung volume (mL) 4175 4195 0.68
FRC lung volume (mL) 1470 1647 0.44
Mean lung density
at TLC (HU)
820 ± 34 823 ± 33 0.13
Air trapping (%) (FRC) 4.6 ± 8.3 7.9 ± 11.8 0.10
Airway luminal area
(TLC) mm
44.7 ± 10.3 45.1 ± 8.5 0.07
Large 103 ± 13 102.9 ± 16 0.73
Medium 30.9 ± 1.8 32 ± 4.5 0.04
Small 13.1 ± 1.6 14.3 ± 3.4 0.01
Airway wall thickness
(fraction luminal diameter)
0.57 ± 0.03 0.57 ± 0.02 0.82
Large 0.45 ± 0.02 0.46 ± 0.03 0.12
Medium 0.58 ± 0.02 0.58 ± 0.03 0.96
Small 0.67 ± 0.02 0.66 ± 0.04 0.03
Table 6 Change in oxidative stress gene expression related to
changes in Forced Expiratory Volume in 1 second (FEV
response to single dose MCh challenge (mean ± SD). P-value for
pre vs. post sulforaphane
Gene Expression (% change from baseline)
Decreased FEV
to MCh
6.11 ± 27.0 73.1 ± 255.4 19.2 ± 40.6
p = 0.67 p = 0.23 p = 0.03
Increased FEV
to MCh
11.8 ± 56.0 141.9 ± 283.7 32.4 ± 75.0
p = 0.38 p = 0.047 p = 0.58
Brown et al. Respiratory Research (2015) 16:106 Page 9 of 12
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Few studies have examined the consumption of foods
rich in antioxidants in general and sulforaphane specific-
ally, on airway reactivity and asthma. Early studies sug-
gested that changes in the diets of children was a
determinant of the worldwide increases in asthma and
allergies [44], and that infrequent consumption of fresh
fruit was associated with impaired lung function [45].
More recently, intake of some vegetables was also asso-
ciated with reduced prevalence of adult asthma [46].
Commonly consumed fruits, vegetables, and nuts, and
high adherence to a traditional Mediterranean diet dur-
ing childhood were shown to have beneficial effects on
symptoms of asthma and rhinitis [47]. Adherence to a
Mediterranean diet during pregnancy also had a protect-
ive effect against asthma-like symptoms and atopy in
childhood [48]. An intervention that administered a food
plant, (Moringa oleifera) known to be rich in glucosino-
lates similar to the precursor of sulforaphane, to 20 asth-
matic subjects daily for 3 weeks, significantly improved
symptom severity, which was accompanied by improve-
ments in FEV
, FVC, and peak expiratory flow rate [49].
Most impressively, a new meta analysis of 12 cohort, 4
population-based casecontrol, and 26 cross-sectional
studies came to the conclusion that reduced risk of
asthma in adults and children was associated with higher
intake of fruit and vegetables (RR = 0.54; 95 % CI, 0.41-
0.69) [50].
The relationship between DI, airway mechanics, and air-
way hyperresponsiveness has been a focus of asthma re-
search for over 2 decades [24]. Several earlier studies have
shown that DI has beneficial effects on human airways
[51, 52]. In healthy individuals, DI taken before exposure to
MCh protected the airways from bronchoconstriction [53].
This property is largely lost in asthma, even in mild disease
(5). Deep inspirations can also reverse airway obstruction
that has been experimentally induced with a direct spasmo-
gen [54]. The bronchodilatory effect of DI is minimally af-
fected in mild asthmatics (5), but decreases with increasing
severity of asthma [55], raising the possibility that the
impairment of this physiologic function of the lung is one
of the determinants of severe obstructive disease.
Our findings are consistent with previous demonstra-
tions that BP and BD effects of DI become more prom-
inent when the MCh-induced bronchoconstriction in
the absence of DI is of substantial magnitude [25]. Our
findings are also consistent with the dependence of the
effects of DI on the magnitude of bronchoconstriction
(Fig. 2). When the responses of our subjects were evalu-
ated by group: (subjects with either a decrease or an in-
crease in response to MCh), the BD and BP outcomes
were dramatically different (Table 3). To our knowledge,
this is the first demonstration of a decrease in response
to MCh leading to a worsening of the BD and BP re-
sponses and not simply a diminution of the response
amplitude [25]. Why this occurred and whether the ef-
fect of sulforaphane on the oxidative stress balance was
involved will require further exploration.
Among nonsmokers, both BP and BD decreased in
those subjects with a decreased FEV
response to MCh,
(Table 7). In contrast, if they had no change, or any in-
crease in their FEV
responses to MCh, both BD and BP
improved significantly. There were similar trends for
smokers, but the changes in BD for subjects with a re-
duced FEV
response to MCh was significantly worse
than those who had non- or marginal FEV
responses to
MCh. Since changes were of similar magnitude in both
smokers and nonsmokers, the difference in the signifi-
cance of that effect may reflect the lower number of
smokers enrolled in the study. Based on animal studies,
a greater effect of sulforaphane on BD and BP would be
expected in smokers. Stimulation of Nrf2 pathways has
been shown to protect against the harmful effects of
cigarette smoke (which releases large quantities of free
radicals and increases lung levels of reactive oxygen and
nitrogen species (ROS and RNS) in murine models [56].
Among all the subjects, there was a significant de-
crease in specific airway resistance (Table 4) with the ad-
ministration of sulforaphane. This is likely explained by
Table 7 BP and BD responses to sulforaphane (SF) arranged according to changes in forced expiratory volume in 1-second (FEV
MCh (mean ± SD). A. Non-smokers (n = 30), B. Smokers (n = 15).
A. Non-smokers
BP (% change) BD (% change)
Before SF After SF p for SF effect Before SF After SF p for SF effect
Decreased FEV
response to MCh 40.4 ± 40.4 3.8 ± 46.8 0.002 33.7 ± 39.4 23.8 ± 36.8 0.20
Increased FEV
response to MCh 49.3 ± 29.7 23.5 ± 35.8 0.004 17.7 ± 27.0 36.3 ± 17.7 0.04
B. Smokers
BP (% change) BD (% change)
Before SF After SF p for SF effect Before SF After SF p for SF effect
Decreased FEV
response to MCh 32.3 ± 34.2 5.1 ± 91.1 0.28 47.1 ± 21.8 22.1 ± 33.4 0.06
Increased FEV
response to MCh 17.1 ± 60.7 48.1 ± 20.6 0.44 22.3 ± 23.1 27.4 ± 20.1 0.63
Brown et al. Respiratory Research (2015) 16:106 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
the concomitant increase in small and medium airway
luminal area (Table 5) after sulforaphane administration.
While these changes did not translate into an improve-
ment in the more commonly used lung function mea-
surements such as FEV
, FVC, and FEV
/FVC, it does
suggests an overall beneficial effect of sulforaphane on
the airways of asthmatic individuals.
In conclusion, administration of sulforaphane, a potent
upregulator of genes protecting against oxidative stress
and inflammation, may be useful both to determine the
mechanisms that lead to asthma, as well as suggesting a
potential therapy to treat asthma. Sulforaphane adminis-
tration improved the BP response in asthmatics who had
an increase in NQO1 gene expression and did not have a
decrease in their initial response to the MCh challenge.
Therefore, sulforaphane administration was able to im-
prove a major defect of even mild asthma. This should en-
courage further examination of this major cytoprotective
signaling pathway as a potential mechanistic approach to
the treatment or prevention of asthma. Furthermore, since
the administration of anti-inflammatory drugs do not
completely prevent the symptoms or progression of the
disease in a substantial subset of asthmatics, these findings
also raise the possibility of the use of sulforaphane, or
foods rich in sulforaphane, as potential adjuvant treat-
ments for asthma.
BD: Bronchodilation; BP: Bronchoprotection; BSE: Broccoli sprout extract;
DI: Deep inspiration; ERV: Expiratory reserve volume; FEV
: Forced expiratory
volume in 1 second; FRC: Functional residual capacity; GST: Glutathione
transferase; GCLM: Glutamine-cysteine ligase, regulatory subunit; IC: Inspiratory
capacity; MAPK: Mitogen-activated protein kinase; MCh: Methacholine;
NfκB: Nuclear factor kappa-light-chain-enhancer of activated B cells;
NQO1: NAD(P)H, Nicotinamide-quinone oxidoreductase 1; Nrf2: Nuclear factor
erythroid-related factor 2; PBMC: Peripheral blood mononuclear cells;
: Provocative concentration causing a 20 % drop in FEV
; RV: Residual
volume; sRAW: specific airway resistance; SVC: Slow vital capacity; TGV: Thoracic
gas volume; TLC: Total lung capacity; TVC: Total vital capacity.
Competing interests
The authors declare that they have no competing interests.
RHB designed the study and analyzed the data. RHB, PT, and JWF wrote the
paper. JWF and PT prepared the sulforaphane-rich broccoli sprout extract.
RHB, CR and AB carried out the clinical and laboratory studies. All authors
read and approved the final manuscript.
Author details
Department of Anesthesiology and Critical Care Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD, USA.
Division of Pulmonary
Medicine and Critical Care, Department of Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD, USA.
Department of
Radiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Lewis B. and Dorothy Cullman Chemoprotection Center, Department of
Pharmacology and Molecular Sciences, Johns Hopkins University School of
Medicine, Baltimore, MD, USA.
Department of Environmental Health
Sciences, Johns Hopkins University School of Public Health, Room E7614,
615 N. Wolfe Street, Baltimore, MD 21205, USA.
Center for Human Nutrition,
Department of International Health, Johns Hopkins University School of
Public Health, Baltimore, MD, USA.
Received: 19 May 2015 Accepted: 21 July 2015
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... Sulforaphane has been widely investigated in numerous human models of disease including cancer chemoprevention, metabolic disorders, and neurological conditions [9][10][11]. Further, SFN has been studied in numerous preclinical and clinical models of lung damage and airway diseases [12][13][14][15][16][17]. The lack of definitive findings from clinical studies to date most likely reflects issues with extract preparations and dosage regimes [18][19][20]. ...
... Likewise, LSF significantly reduced the expression of α-SMA in myofibroblasts in the lamina propria, attenuated the expression of Caveolin-1 induced by OVA, and significantly increased the expression of phosphorylated Smad2 (Fig. 5). In accordance with previous studies, our findings indicated that LSF protected from MCh-induced increases in airway resistance both in vivo (Fig. 2), and ex vivo (Fig. 3) [12]. ...
... The antioxidant effects of LSF involving activation of phase II detoxification pathways have been widely investigated [12]. Additionally, the molecular mechanisms Representative immunofluorescent photomicrographs of lung sections stained with nuclear factor erythroid 2-related factor 2 (Nrf2) (red) show strong staining in ovalbumin-L-sulforaphane (OVA-LSF) mice in both the prevention and reversal model (A). ...
Full-text available
Sulforaphane has been investigated in human pathologies and preclinical models of airway diseases. To provide further mechanistic insights, we explored L-sulforaphane (LSF) in the ovalbumin (OVA)-induced chronic allergic airways murine model, with key hallmarks of asthma. Histological analysis indicated that LSF prevented or reversed OVA-induced epithelial thickening, collagen deposition, goblet cell metaplasia, and inflammation. Well-known antioxidant and anti-inflammatory mechanisms contribute to the beneficial effects of LSF. Fourier transform infrared microspectroscopy revealed altered composition of macromolecules, following OVA sensitization, which were restored by LSF. RNA sequencing in human peripheral blood mononuclear cells highlighted the anti-inflammatory signature of LSF. Findings indicated that LSF may alter gene expression via an epigenetic mechanism which involves regulation of protein acetylation status. LSF resulted in histone and α-tubulin hyperacetylation in vivo, and cellular and enzymatic assays indicated decreased expression and modest histone deacetylase (HDAC) inhibition activity, in comparison with the well-known pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Molecular modeling confirmed interaction of LSF and LSF metabolites with the catalytic domain of metal-dependent HDAC enzymes. More generally, this study confirmed known mechanisms and identified potential epigenetic pathways accounting for the protective effects and provide support for the potential clinical utility of LSF in allergic airways disease.
... 3,4 NRF2-knockout mice exhibit high susceptibility to several oxidative stress-driven diseases such as COPD, 5 asthma, 6 sepsis, 7 and neurological diseases. 8,9 Several preclinical 2,10-12 and few clinical studies [13][14][15] have demonstrated that supplementation of pharmacological activators of NRF2 induces resilience against environmental diseases by mitigating oxidative stress, inflammation, and tissue damage. 12 Short-term and long-term administration of synthetic triterpenoids-CDDO-Im, and CDDO-Me, the most potent NRF2 activators, has been shown to protect from endotoxemia, 10 ischemia-reperfusion injury in the kidney 16 and retina, 17 high-fat induced metabolic syndrome, 18 liver injury, 19 cigarette smoke-induced emphysema, 20 Alzheimer's disease 21 and Parkinson's disease. ...
... Broccoli sprout extract (BSE) containing glucoraphanin (glucosinolate) and sulforaphane (ITC) is the most widely characterized nutraceutical as an NRF2 activator in preclinical and human studies. 23 Supplementation of BSE as a freeze-dried powder in a capsule form improved blood glucose control in diabetic patients, 13 reduced asthmatics symptoms, 14 and reduced autistic spectrum disorders. 24 Intake of BSE in the form of cold beverages increased the detoxification of air pollutants among human subjects in China. ...
Pharmacological activation of nuclear factor erythroid 2 related factor 2 (NRF2) provides protection against several environmental diseases by inhibiting oxidative and inflammatory injury. Besides high in protein and minerals, Moringa oleifera leaves contain several bioactive compounds, predominantly isothiocyanate moringin and polyphenols, which are potent inducers of NRF2. Hence, M. oleifera leaves represent a valuable food source that could be developed as a functional food for targeting NRF2 signaling. In the current study, we have developed a palatable M. oleifera leaf preparation (henceforth referred as ME-D) that showed reproducibly a high potential to activate NRF2. Treatment of BEAS-2B cells with ME-D significantly increased NRF2-regulated antioxidant genes (NQO1, HMOX1) and total GSH levels. In the presence of brusatol (a NRF2 inhibitor), ME-D-induced increase in NQO1 expression was significantly diminished. Pre-treatment of cells with ME-D mitigated reactive oxygen species, lipid peroxidation and cytotoxicity induced by pro-oxidants. Furthermore, ME-D pre-treatment markedly inhibited nitric oxide production, secretory IL-6 and TNF-α levels, and transcriptional expression of Nos2, Il-6, and Tnf-α in macrophages exposed to lipopolysaccharide. Biochemical profiling by LC-HRMS revealed glucomoringin, moringin, and several polyphenols in ME-D. Oral administration of ME-D significantly increased NRF2-regulated antioxidant genes in the small intestine, liver, and lungs. Lastly, prophylactic administration of ME-D significantly mitigated lung inflammation in mice exposed to particulate matter for 3-days or 3-months. In conclusion, we have developed a pharmacologically active standardized palatable preparation of M. oleifera leaves as a functional food to activate NRF2 signaling, which can be consumed as a beverage (hot soup) or freeze-dried powder for reducing the risk from environmental respiratory disease.
... The mechanism underlying the health-promoting effect of sulforaphane relates to its activation of Nrf2/Keap1 signaling pathway [7][8][9][10][11]. This action generally leads to enhanced intracellular antioxidant response and has been reported in human subjects consuming cruciferous vegetables [96][97][98][99][100][101]. Thus, there has been a general interest in understanding the therapeutic effects of sulforaphane against diseases, including diabetes mellitus. ...
... 8 NRF2 deletion causes high susceptibility for various respiratory diseases, including bronchopulmonary dysplasia, respiratory infections, and asthma. 24,25 The developing fetus is especially prone to oxidative stress, and genetic susceptibility resulting from NRF2 polymorphism may have a strong impact on lung development with a high level of PM 10 exposure during the critical window. ...
Background: Exposure to particulate matter (PM) has been known to develop asthma in children and the oxidative stress-related mechanisms are suggested. For the development of asthma, not only the exposure dose but also the critical window and the risk modifying factors should be evaluated. Objective: We investigated whether prenatal exposure to PM10 increases the risk of childhood asthma and evaluated the modifying factors, such as gender and reactive oxidative stress-related gene. Methods: A general population-based birth cohort, the Panel Study of Korean Children (PSKC), including 1572 mother-baby dyads was analyzed. Children were defined to have asthma at age 7 when a parent reported physician-diagnosed asthma. Exposure to PM10 during pregnancy was estimated by land-use regression models based on national monitoring system. TaqMan method was used for genotyping nuclear factor, erythroid 2-related factor, NRF2 (rs6726395). A logistic Bayesian distributed lag interaction model (BDLIM) was used to evaluate the associations between prenatal PM10 exposure and childhood asthma by gender and NRF2. Results: Exposure to PM10 during pregnancy was associated with the development of asthma (aOR 1.03, 95% CI 1.001.06). Stratifying by gender and NRF2 genotype, exposure to PM10 during 26-28 weeks gestation increased the risk of childhood asthma, especially in boys with NRF2 GG genotype. Conclusions: A critical window for PM10 exposure on the development of childhood asthma was during 26-28 weeks of gestation, and this was modified by gender and NRF2 genotype.
... At present, there are a handful of natural compounds that are currently used clinically or undergoing clinical trials for the treatment of diseases, including cancer. For example, Sulforaphane, a naturally occurring isothiocyanate found in high concentration in broccoli, is undergoing clinical trials for the treatment of autism (Singh et al., 2014), asthma (Brown et al., 2015) and Helicobacter pylori infection (Galan et al., 2004). Paclitaxel is a mitotic inhibitor derived from endophytic fungi that grow on the bark of the Pacific yew tree. ...
Ethnopharmacological relevance: Lignosus rhinocerus (Cooke) Ryvarden (also known as Tiger Milk mushroom, TMM), is a basidiomycete belonging to the Polyporaceae family. It has been documented to be used by traditional Chinese physicians and indigenous people in Southeast Asia to treat a variety of illnesses, such as gastritis, arthritis, and respiratory conditions, as well as to restore patients' physical well-being. TMM has also been used in folk medicine to treat cancer. For example, people from the indigenous Kensiu tribe of northeast Kedah (Malaysia) apply shredded TMM sclerotium mixed with water directly onto breast skin to treat breast cancer, while Chinese practitioners from Hong Kong, China prescribe TMM sclerotium as a treatment for liver cancer. L. rhinocerus has previously been demonstrated to possess selective anti-proliferative properties in vitro, however pre-clinical in vivo research has not yet been conducted. Aim of study: This study aimed to examine the anti-tumor activities of L. rhinocerus TM02®, using two different sample preparations [cold water extract (CWE) and fraction] via various routes of administration (oral and intraperitoneal) on an MCF7-xenograft nude mouse model. This study also investigated the inhibitory effect of TM02® CWE and its fractions against COX-2 in vitro using LPS-induced RAW264.7 macrophages, on the basis of the relationship between COX-2 and metastasis, apoptosis resistance, as well as the proliferation of cancer cells. Materials and methods: The first preparation, L. rhinocerus TM02® sclerotium powder (TSP) was dissolved in cold water to obtain the cold water extract (CWE). It was further fractionated based on its molecular weight to obtain the high (HMW), medium (MMW) and low (LMW) molecular weight fractions. The second preparation, known as the TM02® rhinoprolycan fraction (TRF), was obtained by combining the HMW and MMW fractions. TSP was given orally to mimic the daily consumption of a supplement; TRF was administered intraperitoneally to mimic typical tumorous cancer treatment with a rapid and more thorough absorption through the peritoneal cavity. Another experiment was conducted to examine changes in COX-2 activity in LPS-induced RAW264.7 macrophages after a 1-h pre-treatment with CWE, HMW, and MMW. Results: Our results revealed that intraperitoneal TRF-injection (90 μg/g BW) for 20 days reduced initial tumor volume by ∼64.3% (n = 5). The percentage of apoptotic cells was marginally higher in TRF-treated mice vs. control, suggesting that induction of apoptosis as one of the factors that led to tumor shrinkage. TSP (500 μg/g BW) oral treatment (n = 5) for 63 days (inclusive of pre-treatment prior to tumor inoculation) effectively inhibited tumor growth. Four of the five tumors totally regressed, demonstrating the effectiveness of TSP ingestion in suppressing tumor growth. Although no significant changes were found in mouse serum cytokines (TNF-α, IL-5, IL-6 and CCL2), some increasing and decreasing trends were observed. This may suggest the immunomodulatory potential of these treatments that can directly or indirectly affect tumor growth. Pre-treatment with CWE, HMW and MMW significantly reduced COX-2 activity in RAW264.7 macrophages upon 24 h LPS-stimulation, suggesting the potential of L. rhinocerus TM02® extract and fractions in regulating M1/M2 polarization. Conclusion: Based on the findings of our investigation, both the rhinoprolycan fraction and crude sclerotial powder from L. rhinocerus TM02® demonstrated tumor suppressive effects, indicating that they contain substances with strong anticancer potential. The antitumor effects of L. rhinocerus TM02® in our study highlights the potential for further explorations into its mechanism of action and future development as a prophylactic or adjunct therapeutic against tumorous cancer.
... [5][6][7] In the field of chronic lung diseases, few publications had discussed the benefit of SFN in asthma and chronic obstructive pulmonary disease. 8 Furthermore, SFN pretreatment suppressed leukocyte infiltration in human subjects exposed the development of perivascular and alveolar edema within 35 min in ventilated rats. 14 Other physiological abnormalities included elevated wet-to-dry ratio of the isolated lung, composite alterations in bronchoalveolar lavage fluid (BALF), and increased thickness of hyaline membranes. ...
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Background: Owig to the extensive use of mechanical ventilation, risks of acute lung injury are significant in the intensive care unit. Broccoli extract-sulforaphane (SFN) has been investigated as bioactive polyphenol in chronic lung diseases. Aim: The present study aimed to evaluate the preventive effect of SFN in a rat model of ventilator-induced lung injury. Methods: SFN supplement was administrated 30 min before intubation with the dosage of 3 mg/kg. Then, rats were assigned to receive ventilation with a high tidal volume of 40 mL/kg for 6 h, and low ventilation of 6 mL/kg served as controls. Results: The severity of pulmonary edema was mitigated in the SFN-pretreated group with decreased weight ratios of wet to dry lung and total lung to the body, respectively. From bronchoalveolar lavage, SFN treatment suppressed both leukocytes counts and cytokines production. Following ventilator-exerted oxidative burst with the rescue of glutathione level was identified in SFN-pretreated group. Besides, SFN-reduced cell apoptosis was confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and cleavage of caspase-3. Western blotting from lung tissues revealed the upregulation of hemeoxygenase-1 with decreased nuclear factor κB and p38 phosphorylation in SFN-treated group. Conclusion: Our results elucidated the prophylaxis of broccoli extract-SFN could attenuate ventilator-induced oxidative stress, inflammation reaction, and pulmonary edema.
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Objective Sepsis related injury has gradually become the main cause of death in non-cardiac patients in intensive care units, but the underlying pathological and physiological mechanisms remain unclear. The Third International Consensus Definitions for Sepsis and Septic Shock (SEPSIS-3) definition emphasized organ dysfunction caused by infection. Neutrophil extracellular traps (NETs) can cause inflammation and have key roles in sepsis organ failure; however, the role of NETs-related genes in sepsis is unknown. Here, we sought to identify key NETs-related genes associate with sepsis. Methods Datasets GSE65682 and GSE145227, including data from 770 patients with sepsis and 54 healthy controls, were downloaded from the GEO database and split into training and validation sets. Differentially expressed genes (DEGs) were identified and weighted gene co-expression network analysis (WGCNA) performed. A machine learning approach was applied to identify key genes, which were used to construct functional networks. Key genes associated with diagnosis and survival of sepsis were screened out. Finally, mouse and human blood samples were collected for RT-qPCR verification and flow cytometry analysis. Multiple organs injury, apoptosis and NETs expression were measured to evaluated effects of sulforaphane (SFN). Results Analysis of the obtained DEGs and WGCNA screened a total of 3396 genes in 3 modules, and intersection of the results of both analyses with 69 NETs-related genes, screened out seven genes ( S100A12 , SLC22A4 , FCAR , CYBB , PADI4 , DNASE1 , MMP9 ) using machine learning algorithms. Of these, CYBB and FCAR were independent predictors of poor survival in patients with sepsis. Administration of SFN significantly alleviated murine lung NETs expression and injury, accompanied by whole blood CYBB mRNA level. Conclusion CYBB and FCAR may be reliable biomarkers of survival in patients with sepsis, as well as potential targets for sepsis treatment. SFN significantly alleviated NETs-related organs injury, suggesting the therapeutic potential by targeting CYBB in the future.
Dysregulation of innate immune responses can result in chronic inflammatory conditions. Glucocorticoids, the current frontline therapy, are effective immunosuppressive drugs but come with a trade-off of cumulative and serious side effects. Therefore, alternative drug options with improved safety profiles are urgently needed. Sulforaphane, a phytochemical derived from plants of the brassica family, is a potent inducer of phase II detoxification enzymes via nuclear factor-erythroid factor 2-related factor 2 (NRF2) signaling. Moreover, a growing body of evidence suggests additional diverse anti-inflammatory properties of sulforaphane through interactions with mediators of key signaling pathways and inflammatory cytokines. Multiple studies support a role for sulforaphane as a negative regulator of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) activation and subsequent cytokine release, inflammasome activation and direct regulation of the activity of macrophage migration inhibitory factor. Significantly, studies have also highlighted potential steroid-sparing activity for sulforaphane, suggesting that it may have potential as an adjunctive therapy for some inflammatory conditions. This review discusses published research on sulforaphane, including proposed mechanisms of action, and poses questions for future studies that might help progress our understanding of the potential clinical applications of this intriguing molecule.
Objective: Acute liver failure (ALF) is characterized by severe liver dysfunction, rapid progression and high mortality and is difficult to treat. Studies have found that sulforaphane (SFN), a nuclear factor E2-related factor 2 (NRF2) agonist, has anti-inflammatory, antioxidant and anticancer effects, and has certain protective effects on neurodegenerative diseases, cancer and liver fibrosis. This paper aimed to explore the protective effect of SFN in ALF and it possible mechanisms of action. Methods: Lipopolysaccharide and D-galactosamine were used to induce liver injury in vitro and in vivo. NRF2 agonist SFN and histone deacetylase 6 (HDAC6) inhibitor ACY1215 were used to observe the protective effect and possible mechanisms of SFN in ALF, respectively. Cell viability, lactate dehydrogenase (LDH), Fe2+, glutathione (GSH) and malondialdehyde (MDA) were detected. The expression of HDAC6, NRF2, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) were detected by Western blotting and immunofluorescence. Results: Our results show that NRF2 was activated by SFN. LDH, Fe2+, MDA and ACSL4 were downregulated, while GSH, GPX4 and SLC7A11 were upregulated by SFN in vitro and in vivo, indicating the inhibitory effect of SFN on ferroptosis. Additionally, HDAC6 expression was decreased in the SFN group, indicating that SFN could downregulate the expression of HDAC6 in ALF. After using the HDAC6 inhibitor, ACY1215, SFN further reduced HDAC6 expression and inhibited ferroptosis, indicating that SFN may inhibit ferroptosis by regulating HDAC6 activity. Conclusion: SFN has a protective effect on ALF, and the mechanism may include reduction of ferroptosis through the regulation of HDAC6. Please cite this article as: Zhang YQ, Shi CX, Zhang DM, Zhang LY, Wang LW, Gong ZJ. Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity. J Integr Med. 2023; Epub ahead of print.
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Broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can generate the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases (GSTs) and other cytoprotective enzymes. A broccoli sprout-derived beverage providing daily doses of 600 µmol glucoraphanin and 40 µmol sulforaphane was evaluated for magnitude and duration of pharmacodynamic action in a 12-week randomized clinical trial. Two hundred and ninety-one study participants were recruited from the rural He-He Township, Qidong, in the Yangtze River delta region of China, an area characterized by exposures to substantial levels of airborne pollutants. Exposure to air pollution has been associated with lung cancer and cardiopulmonary diseases. Urinary excretion of the mercapturic acids of the pollutants, benzene, acrolein, and crotonaldehyde, were measured before and during the intervention using liquid chromatography tandem mass spectrometry. Rapid and sustained, statistically significant (p ≤ 0.01) increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive compared to the null genotype, irrespective of study arm assignment. Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks.
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Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, also called Nfe2l2) is a transcription factor that regulates the cellular redox status. Nrf2 is controlled through a complex transcriptional/epigenetic and post-translational network that ensures its activity increases during redox perturbation, inflammation, growth factor stimulation and nutrient/energy fluxes, thereby enabling the factor to orchestrate adaptive responses to diverse forms of stress. Besides mediating stress-stimulated induction of antioxidant and detoxification genes, Nrf2 contributes to adaptation by upregulating the repair and degradation of damaged macromolecules, and by modulating intermediary metabolism. In the latter case, Nrf2 inhibits lipogenesis, supports β-oxidation of fatty acids, facilitates flux through the pentose phosphate pathway, and increases NADPH regeneration and purine biosynthesis; observations that suggest it directs metabolic reprogramming during stress.
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The generation of oxidative stress by ambient air pollution particles contributes to the development of allergic sensitization and asthma, as demonstrated by intranasal challenge with well-characterized diesel exhaust particle (DEP) suspensions in humans. This effect is due to the presence of redox active organic chemicals in DEP, and can be suppressed by antioxidants and inducers of phase II enzymes in animals. In this communication, we determined whether the administration of a standardized broccoli sprout extract (BSE), which contains a reproducible amount of the sulforaphane (SFN) precursor, glucoraphanin, could be used to suppress the nasal inflammatory response in human subjects challenged with 300 μg of an aqueous DEP suspension (equivalent to daily PM exposure levels on a Los Angeles freeway). SFN is capable of inducing an antioxidant and phase II response via activation of the nuclear transcription factor (erythroid-derived 2)-like 2 (Nrf2). Previous studies have shown that 70-90% SFN delivered by BSE is absorbed, metabolized, and excreted in humans. An initial intranasal challenge with DEP in 29 human subjects was used to characterize the magnitude of the inflammatory response. Following a 4 week washout, a BSE that delivers a reproducible and standardized dose of 100 μmol SFN in mango juice was administered daily for four days. The nasal DEP challenge was repeated and lavage fluid collected to perform white blood cell (WBC) counts. The average nasal WBC increased by 66% over the initial screening levels and by 85% over the control levels 24 hours after DEP exposure. However, total cell counts decreased by 54% when DEP challenge was preceded by daily BSE administration for 4 days (p < 0.001). Since the SFN dose in these studies is equivalent to the consumption of 100-200 g broccoli, our study demonstrates the potential preventive and therapeutic potential of broccoli or broccoli sprouts rich in glucoraphanin for reducing the impact of particulate pollution on allergic disease and asthma.
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Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane), belonging to a family of natural compounds that are abundant in broccoli, has received significant therapeutic interest in recent years. However, the molecular basis of its effects remains to be elucidated. In this study, we attempt to determine whether sulforaphane regulates the inflammatory response in an ovalbumin (OVA)-induced murine asthma model. Mice were sensitized with OVA, treated with sulforaphane, and then challenged with OVA. Sulforaphane administration significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Additionally, sulforaphane suppressed the increase in the levels of SOCS-3 and GATA-3 and IL-4 expression in the OVA-challenged mice. Collectively, our results demonstrate that sulforaphane regulates Th2 immune responses. This sutdy provides novel insights into the regulatory role of sulforaphane in allergen-induced Th2 inflammation and airway responses, which indicates its therapeutic potential for asthma and other allergic diseases.
Significance Autism spectrum disorder (ASD), encompassing impaired communication and social interaction, and repetitive stereotypic behavior and language, affects 1–2% of predominantly male individuals and is an enormous medical and economic problem for which there is no documented, mechanism-based treatment. In a placebo-controlled, randomized, double-blind clinical trial, daily oral administration for 18 wk of the phytochemical sulforaphane (derived from broccoli sprouts) to 29 young men with ASD substantially (and reversibly) improved behavior compared with 15 placebo recipients. Behavior was quantified by both parents/caregivers and physicians by three widely accepted measures. Sulforaphane, which showed negligible toxicity, was selected because it upregulates genes that protect aerobic cells against oxidative stress, inflammation, and DNA-damage, all of which are prominent and possibly mechanistic characteristics of ASD.
Major bibliographic databases were searched for studies examining the relationship between fruit and vegetable consumption and the risk of wheezing and asthma. Random-effects models were used to pool study results. Subgroup analyses were conducted by fruit and vegetable categories, study design, and age group. Twelve cohorts, 4 population-based case-control studies, and 26 cross-sectional studies published between January 1990 and July 2013 were identified. For the meta-analysis of adults and children, the relative risk (RR) and confidence intervals (CI) when comparing the highest intake group with the lowest intake group were 0.78 (95%CI, 0.70-0.87) for fruit and 0.86 (95%CI, 0.75-0.98) for vegetables. High intake of fruit and vegetables (RR = 0.76; 95%CI, 0.68-0.86 and RR = 0.83; 95%CI, 0.72-0.96) reduced the risk of childhood wheezing. Total intake of fruit and vegetables had a negative association with risk of asthma in adults and children (RR = 0.54; 95%CI, 0.41-0.69). Consuming fruit and vegetables during pregnancy had no association with the risk of asthma in offspring. High intake of fruit and vegetables may reduce the risk of asthma and wheezing in adults and children.
Cancer chemoprevention refers to the use of drugs or natural compounds (e.g., phytochemicals) to prevent cancer. Michael Sporn, who was instrumental in developing the field of chemoprevention, frames the development of cancer as a continuum, likening its latent, frequently invisible development to a smoldering barn full of hay that, before it bursts into flames, is not a safe place to be. In a recent discussion of chemoprevention he describes it as “the arrest or reversal of the progression of premalignant cells towards full malignancy, using physiological mechanisms that do not kill healthy cells” [15]. Stephen Hecht, another pioneer in the field, further suggests that we need to target susceptible individuals for interventions “including chemoprevention using nontoxic or dietary agents with demonstrated efficacy” and avoid “fleeting, flamboyant approaches” in favor of dealing “with lifestyle factors that link cause and prevention” [16]. The last 25 years have provided an abundance of quantifiable evidence to underpin a concept already supported by an ever-more-robust body of epidemiologic evidence—that specific diets can reduce the risks of and protect against cancer and other chronic diseases. The novelty of this approach is rooted in the concept that ingesting certain phytochemicals from specific plants can boost the intrinsic defensive mechanisms of cells that protect against oxidative damage, inflammation, and DNA-damaging chemicals—some of the fundamental causes of chronic disease and aging [17-18]. There are a number of questions specific to the target populations that will need to be addressed in delivering these interventions. We have discussed them previously [19-21] but they bear repeating: Are there countervailing health or ecological risks associated with the intervention? What is to be the delivery vehicle (fresh food or processed food or drink products)? Can the food product or intervention be manufactured or grown locally and sustainably? Can farmers or consumers afford the costs? Is the intervention culturally appropriate? Are there any contraindications? Can the actual cost effectiveness be determined as the intervention is implemented? And finally, will people comply? Adoption of healthier diets is of course an uphill battle, and the effect size will likely be small if efforts to encourage more healthy eating are not based on sound science, but progress is being made. Approaches were recently reviewed in a special issue of the journal Science [22-24].
Sulforaphane is a promising agent under preclinical evaluation in many models of disease prevention. This bioactive phytochemical affects many molecular targets in cellular and animal models; however, amongst the most sensitive is Keap1, a key sensor for the adaptive stress response system regulated through the transcription factor Nrf2. Keap1 is a sulfhydryl-rich protein that represses Nrf2 signaling by facilitating the polyubiquitination of Nrf2, thereby enabling its subsequent proteasomal degradation. Interaction of sulforaphane with Keap1 disrupts this function and allows for nuclear accumulation of Nrf2 and activation of its transcriptional program. Enhanced transcription of Nrf2 target genes provokes a strong cytoprotective response that enhances resistance to carcinogenesis and other diseases mediated by exposures to electrophiles and oxidants. Clinical evaluation of sulforaphane has been largely conducted by utilizing preparations of broccoli or broccoli sprouts rich in either sulforaphane or its precursor form in plants, a stable thioglucose conjugate termed glucoraphanin. We have conducted a series of clinical trials in Qidong, China, a region where exposures to food- and air-borne carcinogens has been considerable, to evaluate the suitability of broccoli sprout beverages, rich in either glucoraphanin or sulforaphane or both, for their bioavailability, tolerability, and pharmacodynamic action in population-based interventions. Results from these clinical trials indicate that interventions with well characterized preparations of broccoli sprouts may enhance the detoxication of aflatoxins and air-borne toxins, which may in turn attenuate their associated health risks, including cancer, in exposed individuals.