Alcohol use disorders (AUDs), including alcohol abuse and dependence, have been linked to the development of acute lung injury (ALI). Prior clinical investigations suggested an association between AUDs and abnormal alveolar epithelial permeability mediated through pulmonary oxidative stress that may partially explain this relationship. We sought to determine if correcting pulmonary oxidative stress in the setting of AUDs would normalize alveolar epithelial permeability in a double-blinded, randomized, placebo-controlled trial of Protandim, a nutraceutical reported to enhance antioxidant activity. We randomized 30 otherwise healthy AUD subjects to receive directly observed inpatient oral therapy with either Protandim (1,350 mg/day) or placebo. Subjects underwent bronchoalveolar lavage (BAL) and blood sampling before study drug administration and after 7 days of therapy; all AUD subjects completed the study protocol without adverse events. BAL total protein was measured at each timepoint as an indicator of alveolar epithelial permeability. In subjects with AUDs, before study drug initiation, BAL total protein values were not significantly higher than in 11 concurrently enrolled controls (P = 0.07). Over the 7-day study period, AUD subjects did not exhibit a significant change in BAL total protein, regardless of their randomization to Protandim {n = 14,-2%[intraquartile range (IQR),-56-146%]} or to placebo [n = 16, 77%(IQR -20-290%); P = 0.19]. Additionally, among those with AUDs, no significant changes in BAL oxidative stress indexes, epithelial growth factor, fibroblast growth factor, interleukin-1β, or interleukin-10 were observed regardless of drug type received. Plasma thiobarbituric acid reactive substances, a marker of lipid peroxidation, decreased significantly over time among AUD subjects randomized to placebo (P < 0.01). These results suggest that Protandim for 7 days in individuals with AUDs who are newly abstinent does not alter alveolar epithelial permeability. However, our work demonstrates the feasibility of safely conducting clinical trials that include serial bronchoscopies in a vulnerable population at risk for acute lung injury.

Oxidative stress has been implicated in the uteroplacental ischemia characteristic of preeclampsia and small-for-gestational-age (SGA) birth, both of which are more common at high (>2500 m) vs low altitude. Since Andeans are protected relative to Europeans from the altitude-associated rise in SGA, we asked whether alterations in maternal antioxidant status or oxidative stress contributed to their protection. Enzymatic antioxidant (erythrocyte catalase and superoxide dismutase [SOD]) activity and a plasma marker of lipid peroxidation (8-iso-PGF2α) were measured during pregnancy and in the non-pregnant state in Andean or European residents of low (400 m) or high altitude (3600-4100 m). Pregnancy and altitude increased catalase and/or SOD activity to a greater extent in Andeans than Europeans. 8-iso-PGF2α levels were independent of altitude and pregnancy. SOD was lower in mothers of SGA infants at weeks 20 and 36. Our findings are consistent with the possibility that elevated enzymatic antioxidant activity contributes to Andean protection against altitude-associated SGA.

For the past 40 years or so, oxidative stress has been increasingly recognized as a contributing factor in aging and in various forms of pathophysiology generally associated with aging. Our view of oxidative stress has been largely "superoxide-centric", as we focused on the pathological sources of this oxygen-derived free radical and the types of molecular havoc it can wreak, as well as on the protection provided by the antioxidant enzymes, especially the superoxide dismutases, catalases, and glutathione peroxidases. In the last decade our view of oxidative stress has broadened considerably, and it is now often seen as an imbalance that has its origins in our genes, and the ways in which gene expression is regulated. At the center of this new focus is the transcription factor called nuclear factor (erythroid-derived 2)-like 2, or Nrf2. Nrf2 is referred to as the "master regulator" of the antioxidant response, modulating the expression of hundreds of genes, including not only the familiar antioxidant enzymes, but large numbers of genes that control seemingly disparate processes such as immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior. Thus, the dysregulation of Nrf2-regulated genes provides a logical explanation for the connections, both direct and indirect, between observable oxidative stress and perhaps 200 human diseases involving these various physiological processes, each reflecting a network involving many gene products. The evolutionary self-association of these many genes under the common control of Nrf2 suggests that the immune and inflammatory systems may present the largest demand for increased antioxidant protection, apart from constitutive oxidative stress resulting from mitochondrial oxygen consumption for metabolic purposes. Gene expression microarray data on human primary vascular endothelial cells and on the SK-N-MC human neuroblastoma-derived cell line have been obtained in response to the dietary supplement Protandim, a potent composition of highly synergistic phytochemical Nrf2 activators. Pathway analysis of results shows significant modulation by Protandim of pathways involving not only antioxidant enzymes, but of those related to colon cancer, cardiovascular disease, and Alzheimer disease.

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways.

Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~3-fold increase in proliferation and ~3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O(2)(•-)) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect.

Therapeutic options for Duchenne muscular dystrophy (DMD), the most common and lethal neuromuscular disorder in children, remain elusive. Oxidative damage is implicated as a pertinent factor involved in its pathogenesis. Protandim((R)) is an over-the-counter supplement with the ability to induce antioxidant enzymes. In this study we investigated whether Protandim((R)) provided benefit using surrogate markers and functional measures in the dystrophin-deficient (mdx)mouse model of DMD. Male 3-week-old mdx mice were randomized into two treatment groups: control (receiving standard rodent chow) and Protandim((R))-supplemented standard rodent chow. The diets were continued for 6-week and 6-month studies. The endpoints included the oxidative stress marker thiobarbituric acid-reactive substances (TBARS), plasma osteopontin (OPN), plasma paraoxonase (PON1) activity, H&E histology, gadolinium-enhanced magnetic resonance imaging (MRI) of leg muscle and motor functional measurements. The Protandim((R)) chow diet in mdx mice for 6 months was safe and well tolerated. After 6 months of Protandim((R)), a 48% average decrease in plasma TBARS was seen; 0.92 nmol/mg protein in controls versus 0.48 nmol/mg protein in the Protandim((R)) group (p =.006). At 6 months, plasma OPN was decreased by 57%(p =.001) in the Protandim((R))-treated mice. Protandim((R)) increased the plasma antioxidant enzyme PON1 activity by 35%(p =.018). After 6 months, the mdx mice with Protandim((R)) showed 38% less MRI signal abnormality (p =.07) than mice on control diet. In this 6-month mdx mouse study, Protandim((R)) did not significantly alter motor function nor histological criteria.

Protandim, a well defined dietary combination of 5 well-established medicinal plants, is known to induce endogenous antioxidant enzymes, such as manganese superoxide dismutase (MnSOD). Our previous studies have shown through the induction of various antioxidant enzymes, products of oxidative damage can be decreased. In addition, we have shown that tumor multiplicity and incidence can be decreased through the dietary administration of Protandim in the two-stage skin carcinogenesis mouse model. It has been demonstrated that cell proliferation is accommodated by cell death during DMBA/TPA treatment in the two-stage skin carcinogenesis model. Therefore, we investigated the effects of the Protandim diet on apoptosis; and proposed a novel mechanism of chemoprevention utilized by the Protandim dietary combination. Interestingly, Protandim suppressed DMBA/TPA induced cutaneous apoptosis. Recently, more attention has been focused on transcription-independent mechanisms of the tumor suppressor, p53, that mediate apoptosis. It is known that cytoplasmic p53 rapidly translocates to the mitochondria in response to pro-apoptotic stress. Our results showed that Protandim suppressed the mitochondrial translocation of p53 and mitochondrial outer membrane proteins such as Bax. We examined the levels of p53 and MnSOD expression/activity in murine skin JB6 promotion sensitive (P+) and promotion-resistant (P-) epidermal cells. Interestingly, p53 was induced only in P+ cells, not P- cells; whereas MnSOD is highly expressed in P- cells when compared to P+ cells. In addition, wild-type p53 was transfected into JB6 P- cells. We found that the introduction of wild-type p53 promoted transformation in JB6 P- cells. Our results suggest that suppression of p53 and induction of MnSOD may play an important role in the tumor suppressive activity of Protandim.

Mitochondrial free radical formation has been implicated as a potential mechanism underlying degenerative senescence, although human data are lacking. Therefore, the present study was designed to examine if resting and exercise-induced intramuscular free radical-mediated lipid peroxidation is indeed increased across the spectrum of sedentary aging. Biopsies were obtained from the vastus lateralis in six young (26 + or - 6 yr) and six aged (71 + or - 6 yr) sedentary males at rest and after maximal knee extensor exercise. Aged tissue exhibited greater (P < 0.05 vs. the young group) electron paramagnetic resonance signal intensity of the mitochondrial ubisemiquinone radical both at rest (+138 + or - 62%) and during exercise (+143 + or - 40%), and this was further complemented by a greater increase in alpha-phenyl-tert-butylnitrone adducts identified as a combination of lipid-derived alkoxyl-alkyl radicals (+295 + or - 96% and +298 + or - 120%). Lipid hydroperoxides were also elevated at rest (0.190 + or - 0.169 vs. 0.148 + or - 0.071 nmol/mg total protein) and during exercise (0.567 + or - 0.259 vs. 0.320 + or - 0.263 nmol/mg total protein) despite a more marked depletion of ascorbate and uptake of alpha/beta-carotene, retinol, and lycopene (P < 0.05 vs. the young group). The impact of senescence was especially apparent when oxidative stress biomarkers were expressed relative to the age-related decline in mitochondrial volume density and absolute power output at maximal exercise. In conclusion, these findings confirm that intramuscular free radical-mediated lipid peroxidation is elevated at rest and during acute exercise in aged humans.

We recently described a coding mutation (L60F) in the mitochondrial superoxide dismutase (SOD2) gene of the human T-cell leukemia-derived cell line, Jurkat. In cell extracts the L60F mutant enzyme showed unusual inhibition by thiol reagents not seen in wild type enzyme. Here we compare the properties of purified recombinant L60F SOD2 with a previously described SOD2 mutant, I58T. Both mutant proteins display a weakened dimer-dimer interaction and thermal instability at 55 degrees C. Both I58T and L60F lose activity at 37 degrees C in the presence of 5 mM N-ethylmaleimide, whereas the wild type SOD2 does not. Each subunit contains one exposed, reactive cysteine residue at position 196, and a second cysteine residue at 140, which is buried and unreactive in the wild type tetramer. We propose that the mutant enzymes, which exist largely as dimers, allow both cysteine residues to react with thiol reagents. When the cysteine residue at 140 was changed to serine by site-directed mutagenesis, both double mutants I58T/C140S and L60F/C140S lose their increased thiol sensitivity. The evolutionary significance of Cys140 is discussed.

BACKGROUND:-The most important determinant of longevity in pulmonary arterial hypertension is right ventricular (RV) function, but in contrast to experimental work elucidating the pathobiology of left ventricular failure, there is a paucity of data on the cellular and molecular mechanisms of RV failure. Methods and Results-A mechanical animal model of chronic progressive RV pressure overload (pulmonary artery banding, not associated with structural alterations of the lung circulation) was compared with an established model of angioproliferative pulmonary hypertension associated with fatal RV failure. Isolated RV pressure overload induced RV hypertrophy without failure, whereas in the context of angioproliferative pulmonary hypertension, RV failure developed that was associated with myocardial apoptosis, fibrosis, a decreased RV capillary density, and a decreased vascular endothelial growth factor mRNA and protein expression despite increased nuclear stabilization of hypoxia-induced factor-1alpha. Induction of myocardial nuclear factor E2-related factor 2 and heme-oxygenase 1 with a dietary supplement (Protandim) prevented fibrosis and capillary loss and preserved RV function despite continuing pressure overload. Conclusion-These data brought into question the commonly held concept that RV failure associated with pulmonary hypertension is due strictly to the increased RV afterload.