Harmful effects behind the daily supplementation of a fixed vegetarian blend in the rat model.

Fruit and vegetables (FV) have long been considered a panacea against major chronic diseases, including cancer. However, there is no convincing epidemiological, clinical or experimental evidence supporting FV chemopreventive ability. A daily mono-supplementation of lyophilized onion, tomato, peach, black grape or lettuce was compared with the daily combined administration of the same FV (5 a day-like diet). Ten days post-treatment, the phase-I/II xenobiotic metabolizing and antioxidant enzyme activities, protein and mRNA levels were investigated. As a marker of oxidative stress, the level of hydroperoxides was measured in rat serum samples. Here we show that a blend of FV orally administered to rats not only potentially manipulates metabolism but also disrupts systemic oxidative homeostasis. A daily combination of the five servings remarkably down-regulates the catalytic activity, protein and mRNA levels of a cohort of hepatic metabolizing enzymes, suggesting a possible depressed clearance upon exposure to ubiquitous carcinogens. Strikingly, we observed an impairment of antioxidant enzymes with a boost in systemic hydroperoxide levels. Our study identifies new potential factors of cancer risk connected with the persistent consumption of fixed servings of FV, suggesting that dietary guidance should rely on a "daily diversification" of FV.

Hepatitis C virus and host cell nuclear transport machinery: a clandestine affair.

There is growing evidence that factors encoded by cytoplasmic replicating viruses functionally interact with components of the nucleocytoplasmic transport apparatus. They do so either to access the cell nucleus, thus affecting genes expression, or to interfere with nuclear transport functionality, hindering host immune response. Recent studies revealed that the hepatitis C virus (HCV) makes no exception, interacting with the host cell nuclear transport machinery at two different levels. On the one hand, small amounts of both core and NS5A localize within the host cell nucleus during productive infection, modulating gene expression and signaling functions to promote persistent infection. On the other hand, HCV infection causes a profound redistribution of certain nucleoproteins to the close proximity of endoplasmic reticulum membrane-derived viral replication factories, where viral RNA amplification occurs. These nucleoporins are believed to form nuclear pore complex-like structures, as suggested by their ability to recruit nuclear localization sequence-bearing proteins. Thus, both processes are linked to virus-induced persistence and pathogenesis, representing possible targets for the development of novel anti-HCV therapeutics.

Redox-Based Flagging of the Global Network of Oxidative Stress Greatly Promotes Longevity.

Despite more than 50 years of investigations into the free radical theory, the direct role of oxidative stress (OS) in aging and age-related diseases remains unproven. Little progress in identifying antioxidant drugs promoting longevity has been made, likely due to selectivity toward one or few radical species, variable efficacy in vivo, inherent pro-oxidant behavior of such drugs, or lack of synergism with metabolic redox homeostasis. Silencing the wide range of reactive free radicals has a great impact on OS-linked outcomes and age-related disorders. Here we show that an innovative, redox-active, multi-radical-scavenger catalytic drug delays the age-associated decline in physiological processes and markedly prolongs the mean lifespan of the adult freshwater annelids Aeolosoma viride by 170%. This unprecedented extension is associated with a decreased OS status. Consistently, treatment of annelids increases their natural resistance to oxygen-derived damage without affecting mitochondrial respiration or reproductive activity. Conversely, the superoxide dismutase (SOD)-mimetic EUK 134 that we selected as a positive control led to an increase in lifespan of ~50%, the same increase previously observed in nematodes. Our results show that reduction of the global network of OS has a profound impact on aging, prompting the development of a possible redox-based therapeutic intervention to counteract the progression of aging.

The roles of hepatitis B virus-encoded X protein in virus replication and the pathogenesis of chronic liver disease.

INTRODUCTION: Hepatitis B virus (HBV) is a major cause of chronic liver disease (CLD) and hepatocellular carcinoma (HCC) worldwide. More than 350 million people are at risk for HCC, and with few treatment options available, therapeutic approaches to targets other than the virus polymerase will be needed. This review suggests that the HBV-encoded X protein, HBx, would be an outstanding target because it contributes to the biology and pathogenesis of HBV in three fundamental ways. AREAS COVERED: First, HBx is a trans-activating protein that stimulates virus gene expression and replication, thereby promoting the development and persistence of the carrier state. Second, HBx partially blocks the development of immune responses that would otherwise clear the virus, and protects infected hepatocytes from immune-mediated destruction. Thus, HBx contributes to the development of CLD without virus clearance. Third, HBx alters patterns of host gene expression that make possible the emergence of HCC. The selected literature cited is from the National Library of Medicine (Pubmed and Medline). EXPERT OPINION: Understanding the mechanisms, whereby HBx supports virus replication and promotes pathogenesis, suggests that HBx will be an important therapeutic target against both virus replication and CLD aimed at the chemoprevention of HCC.

ADAR enzyme and miRNA story: a nucleotide that can make the difference.

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20-23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3' UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Atherosclerosis: lessons from LXR and the intestine.

Modulation of the cholesterol-sensing liver X receptors (LXRs) and their downstream targets has emerged as promising therapeutic avenues in atherosclerosis. The intestine is important for its unique capabilities to act as a gatekeeper for cholesterol absorption and to participate in the process of cholesterol elimination in the feces and reverse cholesterol transport (RCT). Pharmacological and genetic intestine-specific LXR activation have been shown to protect against atherosclerosis. In this review we discuss the LXR-targeted molecular players in the enterocytes as well as the intestine-driven pathways contributing to cholesterol homeostasis with therapeutic potential as targets in the prevention and treatment of atherosclerosis..

Glucocorticoid receptor-mediated transcriptional regulation of N-acetyltransferase 1 gene through distal promoter.

Human arylamine N-acetyltransferase 1, (HUMAN)NAT1, is a phase II xenobiotic-metabolizing enzyme that plays an important role in drug and carcinogen biotransformation and cancer development. Its gene expression has been shown to be regulated by environmental factors. The purpose of the current study is to determine the involvement of nuclear receptors in transcriptional regulation of (HUMAN)NAT1 gene. We show that among the nuclear receptors examined, including the glucocorticoid receptor, retinoid acid receptor-related orphan receptor alpha, constitutive androstane receptor, pregnane X receptor, aryl hydrocarbon receptor, and retinoic acid receptor, the glucocorticoid receptor plays a dominant role in regulating (HUMAN)NAT1 gene expression through distal promoter (P3). The involvement of the glucocorticoid receptor in transcription regulation of (HUMAN)NAT1 gene expression was demonstrated by dexamethasone treatment, reporter assay using plasmid-containing 3 kbp of 5'-end region of promoter 3, and treatment of anti-glucocorticoid RU486 in primary culture of human hepatocytes and transfected HepG2 cells. In addition, translation inhibition did not affect dexamethasone-induced gene expression through P3, suggesting that dexamethasone effect is directly mediated by glucocorticoid receptor activation. Furthermore, deletion analysis revealed the presence of multiple responsive elements within the 3 kbp fragment of P3. Transfection assays in mice using hydrodynamics-based procedure and reporter gene assay in a mouse cell line revealed that glucocorticoid-induced NAT gene expression is species dependent. Dexamethasone treatment of transfected mice and mouse cell line decreased (MOUSE)Nat2 gene expression, (HUMAN)NAT1 homologue. These results suggest that glucocorticoids serve as a modulator for (HUMAN)NAT1 gene expression via the P3-containing 5'-flanking region.

Hydrodynamic gene delivery and its applications in pharmaceutical research.

Hydrodynamic delivery has emerged as the simplest and most effective method for intracellular delivery of membrane-impermeable substances in rodents. The system employs a physical force generated by a rapid injection of large volume of solution into a blood vessel to enhance the permeability of endothelium and the plasma membrane of the parenchyma cells to allow delivery of substance into cells. The procedure was initially established for gene delivery in mice, and its applications have been extended to the delivery of proteins, oligo nucleotides, genomic DNA and RNA sequences, and small molecules. The focus of this review is on applications of hydrodynamic delivery in pharmaceutical research. Examples are provided to highlight the use of hydrodynamic delivery for study of transcriptional regulation of CYP enzymes, for establishment of animal model for viral infections, and for gene drug discovery and gene function analysis.

Nonviral gene transfer as a tool for studying transcription regulation of xenobiotic metabolizing enzymes.

Numerous xenobiotic metabolizing enzymes are regulated by nuclear receptors at transcriptional level. The challenge we currently face is to understand how a given nuclear receptor interacts with its xenobiotics, migrates into nucleus, binds to the xenobiotic response element of a target gene, and regulates transcription. Toward this end, new methods have been developed to introduce the nuclear receptor gene into appropriate cells and study its activity in activating reporter gene expression under the control of a promoter containing xenobiotic response elements. The goal of this review is to critically examine the gene transfer methods currently available. We concentrate on the gene transfer mechanism, advantages and limitations of each method when employed for nuclear receptor-mediated gene regulation studies. It is our hope that the information provided highlights the importance of gene transfer in studying the mechanisms by which our body eliminates the potentially harmful substances and maintains the homeostasis.

The novel radical scavenger IAC is effective in preventing and protecting against post-ischemic brain damage in Mongolian gerbils.

The removal of pathologically generated free radicals produced during ischemia, reperfusion and intracranical hemorrhage seems to be a viable approach to neuroprotection. However, at present, no neuroprotective agent has proven effective in focal ischemic stroke phase III trials, despite the encouraging data in animal models. This study aimed to explore the effect of the brain penetrant low molecular weight radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) in neurological damage subsequent to ischemia-reperfusion injury in Mongolian gerbils. We examined the intraperitoneal effects of IAC on temporary bilateral common carotid artery occlusion (BCCO) by means of morphological and histological analysis of the hippocampus. Significant dose-dependent protective effects of IAC (1 to 10mg/kg b.w.) against neuropathological and morphological brain changes were seen when administered i.p. 1h before temporary BCCO in Mongolian gerbils. When administered up to 6h after BCCO, IAC actually reverses the neurodegenerative processes (e.g. hippocampal cell viability) induced by ischemia in a dose-dependent fashion. Data show that IAC is highly effective in protecting and preventing oxidative brain damage associated with cerebral flow disturbances. It is also effective even in late treatment of the insult, emphasizing its potential role for the management of ischemic stroke patients.

The non-peptidyl low molecular weight radical scavenger IAC protects human pancreatic islets from lipotoxicity.

BACKGROUND: Chronic exposure to high free fatty acids (FFA) can lead to irreversible damage of beta-cell accounting for impaired insulin secretion. Multiple mechanisms concur in generating the damage, but activation of oxidative stress may contribute to the final toxic effect. To better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 24-h pre-culture with 1.0 mmol/l FFA on the function, survival and mRNA expression of several enzymes involved in the generation and scavenging of reactive oxygen species (ROS). MATERIAL AND METHODS: Human islets, prepared by collagenase digestion and density gradient purification from 9 pancreases of multiorgan donors, were incubated for 24-h in the presence 1.0 mmol/l long-chain mixture (oleate:palmitate, 2:1) FFA, with or without 100 micromol/l IAC, a non-peptidyl low molecular weight radical scavenger. At the end of incubation period, insulin secretion was measured by static incubation, and mRNA expression of insulin, Cu/Zn-SOD, Mn-SOD, Catalase, Glutathione peroxidase (GSH-px) and HO-1 by quantitative Real-Time RT-PCR. Nitrotyrosine levels were determined by an ELISA technique. RESULTS: As compared to control incubation (Ctrl, no FFA), exposure to FFA was associated with impaired insulin release and reduced insulin mRNA expression. The presence of IAC in the incubation medium increased insulin release significantly and prevented changes in mRNA expression. Exposure to FFA was associated with oxidative stress as indicated by a significant accumulation of nitrotyrosine and IAC restrained such an increase. mRNA expression of Cu/Zn-SOD, Mn-SOD, Catalase, GSH-Px, and HO-1 were all modified after FFA exposure. These changes were partially prevented in the presence of IAC. CONCLUSIONS: In human islets 24-h exposure to high FFA causes oxidative stress associated with changes of several enzymes involved in ROS scavenging. These effects were prevented by the use of an antioxidant molecule.

Long-term dietary administration of valproic acid does not affect, while retinoic acid decreases, the lifespan of G93A mice, a model for amyotrophic lateral sclerosis.

Mice bearing the mutated gene for Cu/Zn superoxide dismutase (G93A) are a good model for human amyotrophic lateral sclerosis (ALS). They develop progressive limb paralysis paralleled by loss of motor neurons of the cervical and lumbar spinal cord, which starts at 3-3.5 months of age and ends with death at 4-5 months. Several treatments have been attempted to delay clinical symptoms and to extend lifespan, and some have had modest beneficial effects. One such treatment, based on long-term administration of valproic acid (VPA), resulted in controversial results. We report here that, while dietary supplementation with high VPA dosage slows down motor neuron death, as assessed by measurement of a specific marker for cholinergic neurons in the spinal cord, it has no significant effect on lifespan. Recently, the hypothesis has been put forward that a deficiency of retinoic acid (RA) and its signaling may have a role in ALS. We report that long-term dietary supplementation with RA has no effect on the decrease of the cholinergic marker in the spinal cord, but it significantly shortens lifespan of G93A mice.

Glucoraphasatin and glucoraphenin, a redox pair of glucosinolates of brassicaceae, differently affect metabolizing enzymes in rats.

Brassica vegetables are an important dietary source of glucosinolates (GLs), whose breakdown products exhibit anticancer activity. The protective properties of Brassicaceae are believed to be due to the inhibition of Phase-I or induction of Phase-II xenobiotic metabolizing enzymes (XMEs), thus enhancing carcinogen clearance. To study whether GLs affect XMEs and the role of their chemical structure, we focused on two alkylthio GLs differing in the oxidation degree of the side chain sulfur. Male Sprague-Dawley rats were supplemented (per oral somministration by gavage) with either glucoraphasatin (4-methylthio-3-butenyl GL; GRH) or glucoraphenin (4-methylsulfinyl-3-butenyl GL; GRE), at 24 or 120 mg/kg body weight in a single or repeated fashion (daily for four consecutive days), and hepatic microsomes were prepared for XME analyses. Both GLs were able to induce XMEs, showing different induction profiles. While the inductive effect was stronger after multiple administration of the higher GRH dosage, the single lower GRE dose was the most effective in boosting cytochrome P-450 (CYP)-associated monooxygenases and the postoxidative metabolism. CYP3A1/2 were the most affected isoforms by GRH treatment, whereas GRE induced mainly CYP1A2 supported oxidase. Glutathione S-transferase increased up to approximately 3.2-fold after a single (lower) GRE dose and UDP-glucuronosyl transferase up to approximately 2-fold after four consecutive (higher) GRH doses. In conclusion, the induction profile of these GLs we found is not in line with the chemopreventive hypothesis. Furthermore, the oxidation degree of the side chain sulfur of GLs seems to exert a crucial role on XME modulation.

Thromboembolic events and haematological diseases: a case of stroke as clinical onset of a paroxysmal nocturnal haemoglobinuria.

Some haematological diseases are associated to an increased risk of thromboembolic events. We report a case of paroxysmal nocturnal haemoglobinuria (PNH) in which a cerebrovascular event represented the first clinical manifestation of disease. PNH is associated to thromboembolic events, generally of venous districts often involving unusual locations such as mesenteric vessels, sagittal veins, inferior vena cava and renal veins.To our knowledge arterial thrombotic episodes are rare and the involvement of arterial cerebral vessels is exceptional. Then, our case points out the importance of investigating about haematological disorders in all patients presenting with a stroke, in which the common predisposing conditions are excluded.