Vihas T. Vasu

A dose-response study on the effects of purified lycopene supplementation on biomarkers of oxidative stress.

Objective: While tomato product supplementation, containing antioxidant carotenoids, including lycopene, decreases oxidative stress, the role of purified lycopene as an antioxidant remains unclear. Thus, we tested the effects of different doses of purified lycopene supplementation on biomarkers of oxidative stress in healthy volunteers. Methods: This was a double-blind, randomized, placebo-controlled trial, examining the effects of 8-week supplementation of purified lycopene, on plasma lycopene levels, biomarkers of lipid peroxidation {LDL oxidizability, malondialdehyde & hydroxynonenals (MDA & HNE), urinary F2-isoprostanes}, and markers of DNA damage in urine and lymphocytes. Healthy adults (n = 77, age ≥ 40 years), consumed a lycopene-restricted diet for 2 weeks, and were then randomized to receive 0, 6.5, 15, or 30 mg lycopene/ day for 8 weeks, while on the lycopene-restricted diet. Blood and urine samples were collected at the beginning and end of Week 2 of lycopene-restricted diet, and at end of Week 10 of the study. Results: Independent of the dose, plasma lycopene levels significantly increased in all lycopene supplemented groups versus placebo (p < 0.05). ANOVA revealed a significant decrease in DNA damage by the comet assay (p = 0.007), and a significant decrease in urinary 8-hydroxy deoxoguanosine (8-OHdG) at 8 weeks versus baseline (p = 0.0002), with 30 mg lycopene/day. No significant inter- or intra-group differences were noted for glucose, lipid profile, or other biomarkers of lipid peroxidation at any dose/time point. Conclusions: Thus, purified lycopene was bioavailable and was shown to decrease DNA oxidative damage and urinary 8-OHdG at the high dose.

Genome-Wide Screening of Alpha-Tocopherol Sensitive Genes in Heart Tissue from Alpha-Tocopherol Transfer Protein Null Mice (ATTP−/−)

Alpha‐tocopherol transfer protein (ATTP) null mice (ATTP−/−) have a systemic deficiency of alpha‐tocopherol (AT). The heart AT levels of ATTP−/− are <10% of those in ATTP+/+ mice. The genomic responses of heart to AT deficiency were determined in 3 months old male ATTP−/− mice and compared with their ATTP+/+ littermate controls using Affymetrix 430A 2.0 high density oligonucleotide arrays. Differential analysis of ∼13 000 genes identified repression of genes related to immune system and activation of genes related to lipid metabolism and inflammation with no significant change in the expression of classical antioxidant genes (catalase, superoxide dismutase, glutathione peroxidase) in ATTP−/− as compared to ATTP+/+ mice. The present data identifies novel classes of AT sensitive genes in heart tissue.

Dietary α‐tocopherol and neuromuscular health: Search for optimal dose and molecular mechanisms continues!

Rodents fed alpha-tocopherol (alphaT)-depleted diets develop neuromuscular deficits. Unequivocal role of alphaT in the prevention of these deficits is confounded by possible neurotoxic oxidant products generated, ex vivo in alphaT-depleted diets. The discovery that large doses of alphaT could ameliorate neuromuscular deficits, attributed to very low serum alphaT caused by mutations in either the microsomal triglyceride transfer protein or the alphaT-transfer protein (alphaTTP), underscores the necessity of alphaT for neuromuscular health in humans. The discovery of human alphaTTP provided physiological relevance to biochemical data from rodents documenting alphaT-binding transfer protein, expressed exclusively in liver. The cloning of alphaTTP gene and the creation of alphaTTP-knockout mice allowed to achieve severe systemic alphaT deficiency in brain and muscles, possibly at birth, eliminating the possible confounding effects of ex vivo-generated oxidant products in vitamin E-stripped diets. alphaTTP-knockout mice have proven useful models to discover alphaT-regulated phenotypes and molecular actions of alphaT in vivo. The results suggest that antioxidant and non-antioxidant actions of alphaT in vivo may not be mutually exclusive. These studies also suggest that low levels of dietary alphaT can achieve in excess of nanomolar alphaT levels in tissues and maintain normal neuromuscular functions. This is consistent with biochemical and crystallographic data of alpha-TTP and of other alphaT-binding proteins that have dissociation constants in nanomolar range. Molecular mechanisms that cause a long delay for the development of deficiency symptoms remain enigmatic. It is likely that alphaT is metabolically stable in post-mitotic neurons and myocytes and, if it undergoes redox-cycling in vivo, a large repertoire of alphaT-regenerating systems maintains its biological activity before it is totally depleted.

Mice lacking α-tocopherol transfer protein gene have severe α-tocopherol deficiency in multiple regions of the central nervous system

Ataxia with vitamin E deficiency is caused by mutations in alpha-tocopherol transfer protein (alpha-TTP) gene and it can be experimentally generated in mice by alpha-TTP gene inactivation (alpha-TTP-KO). This study compared alpha-tocopherol (alpha-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and alpha-TTP-KO mice. All brain regions of female WT mice contained significantly higher alpha-T than those from WT males. alpha-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain alpha-T concentrations do not appear to be determined by alpha-TTP expression which was undetectable in all brain regions. All the brain regions of alpha-TTP-KO mice were severely depleted in alpha-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of alpha-TTP-KO mice. The results show that both gender and the hepatic alpha-TTP, but not brain alpha-TTP gene expression are important in determining alpha-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in alpha-TTP-KO mice in spite of the severe alpha-tocopherol deficiency in the brain starting at an early age.

Maternal obesity affects gene expression and cellular development in fetal brains

Abstract Objectives Female rat neonates reared on a high carbohydrate (HC) milk formula developed chronic hyperinsulinemia and adult-onset obesity (HC phenotype). Furthermore, we have shown that fetal development in the HC intrauterine environment (maternal obesity complicated with hyperinsulinemia, hyperleptinemia, and increased levels of proinflammatory markers) resulted in increased levels of serum insulin and leptin in term HC fetuses and the spontaneous transfer of the HC phenotype to the adult offspring. The objectives of this study are to identify changes in global gene expression pattern and cellular development in term HC fetal brains in response to growth in the adverse intrauterine environment of the obese HC female rat. Methods GeneChip analysis was performed on total RNA obtained from fetal brains for global gene expression studies and immunohistochemical analysis was performed on fetal brain slices for investigation of cellular development in term HC fetal brains. Results Gene expression profiling identified changes in several clusters of genes that could contribute to the transfer of the maternal phenotype (chronic hyperinsulinemia and adult-onset obesity) to the HC offspring. Immunohistochemical analysis indicated diminished proliferation and neuronal maturation of stem-like cells lining the third ventricle, hypothalamic region, and the cerebral cortex in HC fetal brains. Discussion These results suggest that maternal obesity during pregnancy could alter the developmental program of specific fetal brain cell-networks. These defects could underlie pathologies such as metabolic syndrome and possibly some neurological disorders in the offspring at a later age.

Sarcolipin and ubiquitin carboxy-terminal hydrolase 1 mRNAs are over-expressed in skeletal muscles of α-tocopherol deficient mice

The transcriptome of ataxic muscles from α-tocopherol transfer protein deficient (ATTP-KO), 23-month old, mice was compared with that of their normal littermates. Genes encoding sarcolipin (sln) and ubiquitin carboxyl-terminal hydrolase (uchl1) were over-expressed (≥10-fold) in ataxic muscles. SLN is a 3.2 kDa membrane protein that binds to sarcoplasmic reticulum calcium ATPase, regulates Ca+ + transport and muscle relaxation–contraction cycles. UCHL1 is a 24.8 kDa member of proteosome proteins; it is over-expressed in myofibrillar myopathy and is associated with neurodegenerative diseases. Furthermore, six additional transcripts, three encoding thin-filament proteins and three encoding Ca+ + sensing proteins that participate in contraction-relaxation cycle, and eight transcripts that encode members of lysosomal proteins were also over-expressed in ataxic muscles. These observations suggest that chronic α-tocopherol (AT) deficiency activates critical genes of muscle contractility and protein degradation pathways, simultaneously. The magnitude of induction of sln and uchl1 was lower in asymptomatic, 8-month old, ATTP-KO mice and in 8-month old mice fed an AT-depleted diet. These studies suggest sln and uchl1 genes as novel targets of AT deficiency and may offer molecular correlates of well documented descriptions of neuromuscular dysfunctions in AT-deficient rodents. Since the neuromuscular deficits of ATTP-KO mice appear to be similar to those of patients with ATTP mutations, it is suggested that over-expression of sln and uchl1 may also contribute to AT-sensitive ataxia in humans.

Severe Vitamin E deficiency modulates airway allergic inflammatory responses in the murine asthma model

Allergic asthma is a complex immunologically mediated disease associated with increased oxidative stress and altered antioxidant defenses. It was hypothesized that α-tocopherol (α-T) decreases oxidative stress and therefore its absence may influence allergic inflammatory process, a pathobiology known to be accompanied by oxidative stress. Therefore, selected parameters of allergic asthma sensitization and inflammation were evaluated following ovalbumin sensitization and re-challenge of α-T transfer protein (TTP) knock-out mice (TTP−/−) that have greatly reduced lung α-T levels (e.g.<5%) compared to their litter mate controls (TTP+/+). Results showed that severe α-T deficiency result in a blunted lung expression of IL-5 mRNA and IL-5 protein and plasma IgE levels compared with TTP+/+ mice following immune sensitization and rechallenge, although lung lavage eosinophil levels were comparable in both genomic strains. It is concluded that the initial stimulation of immune responses by the TTP−/− mice were generally blunted compared to the TTP+/+ mice, thus diminishing some aspects of subsequent allergic inflammatory processes.

Genome wide responses of murine lungs to dietary α-tocopherol

α-tocopherol (α-T) may affect biological processes by modulating mRNA concentrations. This study screened the responses of ∼15,000 lung mRNAs to dietary α-T in mice. The lung was chosen as the target organ because it is subjected to cyclical variations in oxidant and inflammatory stressors and α-T has been implicated in their modulations. The analysis identified ∼400 mRNAs sensitive to α-T status of lungs determined by dietary α-T. The female lung transcriptome appears to be more sensitive to the α-T status than that of the male lungs. Here, we focus on the induction of 13 cytoskeleton genes by dietary α-T because they were similarly induced in the male and the female lungs. Their inductions were confirmed by quantitative-real-time-polymerase chain reaction (qRT-PCR). Immunohistochemical analyses of three of the encoded proteins suggest that they are expressed in lung vasculature and alveolar regions. The data suggest that the lung α-T status may modulate cytoarchitecture of lungs.

Myeloperoxidase-dependent oxidative metabolism of nitric oxide in the cystic fibrosis airway.

BACKGROUND Decreased expired nitric oxide (eNO) is commonly observed in cystic fibrosis (CF) patients and is usually explained by dysregulation of NO synthase (NOS) isoforms in respiratory tract epithelium. Later stages of this disease are accompanied by intense airway infiltration of phagocytes with high NOS activity, abundant levels of the hemoprotein myeloperoxidase (MPO) and significant production of significant reactive oxygen species. METHODS This study characterizes the contribution of the high airway levels of MPO to decreased eNO levels in adult CF patients. NO metabolites (NO(x)) and MPO levels in fresh sputum of control and adult CF patients were determined and related to measurements of eNO and to in vitro consumption of NO in CF sputum. RESULTS Despite essentially equal levels of NO(x) in sputum, eNO was 2- to 3-fold lower in CF patients compared to healthy controls. In CF patients, eNO levels were negatively associated with sputum peroxidase activity. In vivo correlations were confirmed by ex vivo studies of NO consumption by MPO in CF sputum. Immunodepletion studies confirmed MPO as the major heme peroxidase in CF sputum contributing to the hydrogen peroxide (H(2)O(2))-dependent consumption of NO. CONCLUSIONS In CF airways MPO acts as a phagocyte-derived NO oxidase that diminishes NO bioavailability at airway surfaces, possibly identifying this peroxidase as a potential target for therapeutic intervention.

Nr1d1, an Important Circadian Pathway Regulatory Gene, Is Suppressed By Cigarette Smoke in Murine Lungs

Nuclear receptor subfamily 1, group D member 1 (Nr1d1), also known as Rev-erb-α, belongs to the family of “orphan receptors” and functions as a member of clock gene family. In addition to being an important member of clock circuitry, Nr1d1, also regulates cell proliferation, lipid metabolism, and inflammation and is also touted as a tumor suppressor. Our focus on Nr1d1 was stimulated by data from a genome-wide search for mRNA correlates of cigarette smoke (CS) sensitive—whole smoke (WS) and filtered smoke (FS)—lung transcriptomes in tumor-resistant C57BL6 and tumor-susceptible AJ mice strains. Differential analysis of ~15 000 genes using Affymetrix 430A 2.0 high-density oligonucleotide arrays identified modulation of genes related to circadian pathways by CS in lungs of both mouse strains. Nr1d1 expression was downregulated by both WS and FS irrespective of mouse strain as compared to respective air-breathing controls. WS was more effective than FS on decreasing Nr1d1 expression. The present data suggest that transcriptional regulation of Nr1d1 by CS may affect circadian rhythmicity and thus may play a complementary role in CS-induced lung respiratory tract pathobiology and/or lung tumorigenesis.