Extracorporeal blood oxygenation and ozonation: clinical and biological implications of ozone therapy

Abstract

Some lines of evidence have suggested that the challenge to antioxidants and biomolecules provoked by pro-oxidants such as ozone may be used to generate a controlled stress response of possible therapeutic relevance in some immune dysfunctions and chronic, degenerative conditions. Immune and endothelial cells have been proposed to be elective targets of the positive molecular effects of ozone and its derived species formed during blood ozonation. On the bases of these underlying principles and against often prejudicial scepticism and concerns about its toxicity, ozone has been used in autohemotherapy (AHT) for four decades with encouraging results. However, clinical application and validation of AHT have been so far largely insufficient. Latterly, a new and more effective therapeutic approach to ozone therapy has been established, namely extracorporeal blood oxygenation and ozonation (EBOO). This technique, first tested in vitro and then in vivo in sheep and humans (more than 1200 treatments performed in 82 patients), is performed with a high-efficiency apparatus that makes it possible to treat with a mixture of oxygen–ozone (0.5–1 μg/ml oxygen) in 1 h of extracorporeal circulation up to 4800 ml of heparinized blood without technical or clinical problems, whereas only 250 ml of blood can be treated with ozone by AHT. The EBOO technique can be easily adapted for use in hemodialysis also. The standard therapeutic cycle lasts for 7 weeks in which 14 treatment sessions of 1 h are performed. After a session of EBOO, the interaction of ozone with blood components results in 4–5-fold increased levels of thiobarbituric acid reactants and a proportional decrease in plasma protein thiols without any appreciable erythrocyte haemolysis. On the basis of preliminary in vitro evidence, these simple laboratory parameters may represent a useful complement in the routine monitoring of biological compliance to the treatment. The clinical experience gained so far confirms the great therapeutic potential of EBOO in patients with severe peripheral arterial disease, coronary disease, cholesterol embolism, severe dyslipidemia, Madelung disease, and sudden deafness of vascular origin. Extensive investigation on oxidative stress biomarkers and clinical trials are under way to validate this new technique further.     

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert‐butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12–Atg5 conjugate, Atg7 contents decreased but LC3‐II accumulated in tBHP‐treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ‐1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions. J. Cell. Biochem. 114: 212–219, 2012. © 2012 Wiley Periodicals, Inc.     

Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis

Abstract

The production of ROS is an inevitable consequence of metabolism. However, high levels of ROS within a cell can be lethal and so the cell has a number of defences against oxidative cell stress. Occasionally the cell's antioxidant mechanisms fail and oxidative stress occurs. High levels of ROS within a cell have a number of direct and indirect consequences on cell signalling pathways and may result in apoptosis or necrosis. Although some of the indirect effects of ROS are well known, limitations in technology mean that the direct effects of the cell's redox environment upon proteins are less understood. Recent work by a number of groups has demonstrated that ROS can directly modify signalling proteins through different modifications, for example by nitrosylation, carbonylation, di-sulphide bond formation and glutathionylation. These modifications modulate a protein's activity and several recent papers have demonstrated their importance in cell signalling events, especially those involved in cell death/survival. Redox modification of proteins allows for further regulation of cell signalling pathways in response to the cellular environment. Understanding them may be critical for us to modulate cell pathways for our own means, such as in cytotoxic drug treatments of cancer cells. Protein modifications mediated by oxidative stress can modulate apoptosis, either through specific protein modifications resulting in regulation of signalling pathways, or through a general increase in oxidised proteins resulting in reduced cellular function. This review discusses direct oxidative protein modifications and their effects on apoptosis.     

Alleviation of free radical mediated oxidative and genotoxic effects of cadmium by farnesol in Swiss albino mice

Abstract

Farnesol is an isoprenoid found in essential oils of ambrette seeds, citronella and in various aromatic plants. Exposure to cadmium from various sources affects the renal system adversely and Cd is an established genotoxic agent. In the present study, we evaluated the antigenotoxic and antioxidant efficacy of farnesol against cadmium chloride (CdCl₂)-induced renal oxidative stress and genotoxicity in Swiss albino mice. Single, intraperitoneal doses of CdCl₂(5 mg/kg body weight) for 24 h resulted in a significant (P < 0.001) increase in chromosomal aberration and micronuclei formation. The oral administration of farnesol at two doses (1% and 2% per kg body weight) for seven consecutive days showed significant (P < 0.05) suppression of the genotoxic effects of CdCl₂ in the modulator groups. To study the mechanism by which farnesol exerts its antigenotoxic potential, enzymes involved in metabolism and detoxification were estimated. CdCl₂ intoxication adversely affected the renal antioxidant armory and increased TBARS formation and xanthine oxidase levels significantly (P < 0.001). Farnesol showed a significant (P < 0.001) recovery in antioxidant status viz, GSH content (and its dependent enzymes) and catalase activity. Farnesol pretreatment in CdCl₂-intoxicated mice showed marked (P < 0.001) suppression of TBARS' formation and XO activity. Our results support the conclusion that the anticlastogenic effect of farnesol could be due to restoration of antioxidants and inhibition of oxidative damage.     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.