This is the first clinical study to demonstrate that oral sulforaphane can enhance Phase II antioxidant enzyme expression in human airway cells. The induction of Phase II enzymes by SFN and other Nrf-2 activators has previously been shown using in vitro and animal models, 8, 11, 14, 24–25
however this is the first data to clearly demonstrate this biological effect of SFN in vivo in the human airway. Previous investigation has confirmed that Phase II enzyme mRNA expression as measured by RT-PCR correlates with protein and enzyme activity levels. This has been demonstrated with NQO1 protein expression by Western blot in human lymphocytes8
and GST enzyme activity correlation with GSTM1 mRNA expression in normal human bronchial epithelial cells.11
This evidence supports the potential biologic significance of the marked Phase II enzyme mRNA induction now described with SFN administration in human subjects. The up regulation of endogenous Phase II enzymes was accomplished without apparent toxicity using the administration of oral BSH, a natural readily available source of SFN which was well-tolerated by study subjects. This finding may have important therapeutic implications with regard to asthma and other conditions associated with airway OS.
Cellular OS is believed to be an important underlying pathologic mechanism in the inflammation associated with asthma. Such OS may result from the cellular inflammation seen in asthma, but there is strong evidence that environmental respiratory exposures to air pollutants (i.e. diesel exhaust particles, ozone, NO2) and pollen fragments cause OS in the airways which subsequently leads to tissue inflammation and clinical symptoms.3, 26–28
Cellular exposure to pollutant reactive chemicals or pollens generates reactive oxygen species (ROS) capable of oxidizing proteins, lipids, and DNA. This results in subsequent glutathione depletion and the activation of intrinsic cellular antioxidant defenses via Nrf2 signaling.29
A primary mechanism of this pathway is the activation of the antioxidant response element (ARE) and induction of Phase II metabolizing enzymes.30
This group of enzymes includes glutathione transferases (GSTM, GSTP), heme-oxygenase (HO1), and NAD(P)H:quinone oxidoreductase (NQO1) as well as other enzymes that serve to metabolize xenobiotic chemicals and neutralize ROS.31–32
If this cytoprotective antioxidant response is insufficient or overwhelmed by heavy oxidative burden, glutathione depletion continues resulting in MAPK and NFKB signaling and gene transcription for a cascade of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-alpha as well as chemokines and cellular adhesion molecules.33
In addition to these pro-inflammatory effects of environmentally-induced OS, there is compelling evidence that OS also serves as a potent adjuvant to enhance Th2-responses to allergens and may initiate immune sensitization to common environmental allergens.34–36
The recognized importance of OS in respiratory inflammation has spurred recent interest in therapeutic measures to prevent or reduce OS. At present, the effects of current allergy and asthma therapy on OS are unclear.37–41
As current standard treatment may not adequately address respiratory OS burden induced by environmental exposures, additional therapies protecting against OS may represent a significant step in reducing the morbidity of allergic respiratory disease.
Our current investigation explores a unique strategy to enhance an important endogenous cytoprotective response designed to prevent cellular damage from ROS. The Nrf-2 signaling pathway is a negative regulator of inflammation through induction of many antioxidant, cytoprotective, and detoxification enzymes.42
These inducible Phase II enzymes represent an early and sensitive response to OS by scavenging ROS and metabolizing xenobiotics such as air pollutants. SFN, as used in our investigation, has a number of attractive qualities with regard to human use. Glucosinolate precursors of SFN are naturally found in cruciferous vegetables with previous studies demonstrating bioavailability and basic pharmacokinetics with human consumption. A number of human studies have demonstrated the safety and tolerability of glucosinolates and SFN, findings further confirmed by our recent study data.16–19
In vitro, animal, and human studies have supported the beneficial biological effects of SFN, with Phase II enzyme induction believed to be an important mechanism of action for these observed effects.43–46
With regard to respiratory inflammation, recent in vitro investigations have shown Phase II enzyme induction with SFN to be effective in blocking the pro-allergic effects of DEP on human B-cells as DEP-induced IgE enhancement is inhibited by preculture with SFN.8
Additional studies using human BEC confirm this strategy to be protective against DEP extract oxidative effects.11
SFN effectively upregulates Phase II enzyme expression and blocks DEP extract induced IL-8, GM-CSF, and IL-1β production by human BEC. Thus, while the protective antioxidant effects of PII enzyme induction have yet to be established in human clinical studies, compelling data exists to support the potent anti-inflammatory effects of this strategy in the setting of DEP-induced OS.
Our placebo-controlled study is the first to examine the in vivo effects of SFN administration on PII enzyme expression in the human airway. At BSH doses > 100 grams (51 μmol SFN) daily, we observed a significant dose-response effect in Phase II enzyme expression as measured by RT-PCR using cells recovered from nasal lavage. Our NL collection yields predominantly respiratory epithelial cells making such findings congruent with SFN-mediated PII enzyme induction seen in previous studies using human BEC. We did not observe SFN-induced changes in NL cell counts or differentials, yet the increase in mean expression of PII enzymes ranged from 101% for GSTP1 to 199% for NQO1 at the highest dose of BSH administration. This represents a doubling to tripling of baseline enzyme expression rates. Also significant is the observation that measured increases in individual sentinel PII enzymes were strongly associated with increases in all other PII enzymes. This positive linear-correlation is consistent with our current understanding of a common mechanism of induction for these enzymes, i.e. Nrf-2 signaling with resultant ARE activity promoting transcription of numerous PII enzymes.
While our study was not designed to examine the pharmacokinetics (pK) of oral SFN, the results appear to confirm previously described human pK results. SFN has an apparent volume of distribution of 59.9 ± 7.0 L, consistent with total body water, a mean half-life of 1.77 ± 0.13 hours, and first-order kinetics.19
Previous dosing of 200 μmol SFN has yielded peak plasma levels of 2.00 ± 0.30 μmol/l, suggesting 60% bioavailability.17
Our results are consistent with these previous observations. Using the aforementioned pK parameters, the 200 gram BSH dose (102 μmol SFN) used in our study would be expected to give a peak serum concentration of 1.02 μmol/l. Expected serum concentration 24 hours later (14 t1/2) would be 0.0622 nmol/l. For samples collected nearer the 28 hour mark, the expected concentration is 0.0155 nmol/l. Our serum samples analyzed for SFN content 24 ± 4 hours after the 200 gram BSH dose are within this range (0.0115–0.0121 nmol).
This study demonstrating the potent biological effects of oral SFN on PII enzyme expression in the human upper airway provides vital information for planning additional clinical trials. Future human studies will be necessary to thoroughly investigate the potential beneficial effects of Phase II enzyme induction on environmentally-induced oxidative stress and associated allergic airway inflammation. Currently, it is unknown whether the observed increase in PII enzyme expression will be sufficient to prevent or reduce respiratory OS in the human airway. Also unknown is whether higher doses of SFN will lead to further increases in PII enzyme expression or whether toxicity will be dose-limiting. Our experience, and those of other investigators, is that the currently reported doses are well-tolerated and non-toxic. Additionally, genetic polymorphisms affecting PII enzymes will be an important consideration in future studies examining this therapeutic strategy. We excluded GSTM1 null subjects from our analysis so as to have a clear picture of the induction effects of SFN on PII enzymes. However, with regard to the clinical value of SFN, the presence or absence of specific functional enzymes may be quite important in the resulting therapeutic or nominal effect. Given the complex network of PII antioxidant enzymes, gene-gene-environment interactions are likely to have a strong influence on the observed responses to specific antioxidant therapies.47–48
Careful selection of susceptible target populations based on genetic polymorphisms will be an important consideration for future interventional studies aimed at reducing pollutant-induced oxidative stress.
In summary, our placebo-controlled human study has demonstrated that oral SFN contained in BSH can significantly induce PII enzyme expression in the human airway. This data allows for future clinical studies to examine the potential benefit of SFN in abrogating allergic respiratory inflammation from oxidant stimuli and demonstrates proof-of-concept for Nrf2-activation as a mechanism of PII enzyme upregulation in the human respiratory tract.