Ozone oxidative preconditioning: a protection against cellular damage by free radicals.

There is some anecdotal evidence that oxygen-ozone therapy may be beneficial in some human diseases. However so far only a few biochemical and pharmacodynamic mechanisms have been elucidated. On the basis of preliminary data we postulated that controlled ozone administration would promote an oxidative preconditioning preventing the hepatocellular damage mediated by free radicals. Six groups of rats were classified as follows: (1) negative control, using intraperitoneal sunflower oil; (2) positive control using carbon tetrachloride (CCl4) as an oxidative challenge; (3) oxygen-ozone, pretreatment via rectal insufflation (15 sessions) and after it, CCl4; (4) oxygen, as group 3 but using oxygen only; (5) control oxygen-ozone, as group 3, but without CCl4; group (6) control oxygen, as group 5, but using oxygen only. We have evaluated critical biochemical parameters such as levels of transaminase, cholinesterase, superoxide dismutase, catalase, phospholipase A, calcium dependent ATPase, reduced glutathione, glucose 6 phosphate dehydrogenase and lipid peroxidation. Interestingly, in spite of CCl4 administration, group 3 did not differ from group 1, while groups 2 and 4 showed significant differences from groups 1 and 3 and displayed hepatic damage. To our knowledge these are the first experimental results showing that repeated administration of ozone in atoxic doses is able to induce an adaptation to oxidative stress thus enabling the animals to maintain hepatocellular integrity after CCl4 poisoning.     

Ascorbic acid: metabolism and functions of a multi-facetted molecule

Ascorbic acid (vitamin C) is the most abundant antioxidant in plants. Its biosynthetic pathway via GDP-D-mannose and L-galactose, which was proposed only recently, is now supported by molecular genetic evidence from Arabidopsis thaliana and transgenic potato plants. Except for the last step (which is located on the inner mitochondrial membrane) the pathway is cytosolic, sharing GDP-sugar intermediates with cell-wall polysaccharide and glycoprotein synthesis. Ascorbate peroxidase is emerging as a key enzyme in the fine control of H(2)O(2) concentration; its expression being controlled by redox signals and H(2)O(2). Convincing evidence of the involvement of ascorbate in cell division and growth is also accumulating. Its role as a cofactor in the synthesis of cell wall hydroxyproline-rich glycoproteins is one mechanism for this function.     

Ascorbic acid metabolism in geranium and grape

l-Ascorbic acid-[UL-¹⁴C] has been used to follow the appearance of ¹⁴C-labeled oxalic acid and tartaric acid as metabolic products of oxidative cleavage of ascorbic acid in geranium apices (Pelargonium crispum). The enantiomeric specificity of ascorbic acid metabolism was established in geranium by comparing the incorporation of d- and l-ascorbic acid-[6-¹⁴C] in the presence of l-ascorbic acid-[4-³H]. l-Ascorbic acid-[4-³H] has been used to demonstrate the retention of ³H during biosynthesis of l-(+)-tartaric acid in the geranium and its exchange with water during biosynthesis of l-( +)-tartaric acid in the grape.     

Ascorbic acid radicals induced by the action of radiation in tissues from rat organs frozen at 77 K

Subsequent annealing technique and computer assistant analysis of EPR spectra were used to isolate an asymmetric EPR signal Rs(g = 2,0051; delta H = 0.8 mT) from the EPR spectrum of rat spleen gamma-irradiated at 77 K. Radicals with the same EPR spectrum were registered in: 1) water solution of ascorbic acid (2.10(-2) M, pH 3.4) frozen and irradiated at 77 K and 2) water-glycerol solution of ascorbic acid (10(-2) M, pH 10.3) frozen rapidly at the moment of intensive autooxidation. These model experiments allow to conclude that Rs signal is caused by the radicals of semidehydroascorbic acid. Radiochemical yield of these radicals as well as of all the radicals induced by gamma radiation in the whole rat tissues were measured. The EPR signal (Rs) is equivalent to the well known "artifact" signal of lyophilized tissues. The explanation of the mechanism of the radicals formation taking place under annealing of the frozen and irradiated tissues was suggested.     

Microsomal metabolism of ciprofloxacin generates free radicals

Ciprofloxacin (CPFX) is a widely used fluoroquinolone antibiotic with a broad spectrum of activity. However, clinical experience has shown a possible incidence of undesirable adverse effects including gastrointestinal, skin, hepatic, and central nervous system (CNS) functions, and phototoxicity. Several examples in the literature data indicate that free radical formation might play a role in the mechanism of some of these adverse effects, including phototoxicity and cartilage defects. The purpose of this study is to investigate free radical formation during the metabolism of CPFX in hepatic microsomes using electron spin resonance (ESR) spectroscopy and spin trapping technique. We then investigate the effects of a cytochrome P450 inhibitor, SKF 525A, Trolox, and ZnCl2 on CPFX-induced free radical production. Our results show that CPFX induces free radical production in a dose- and time-dependent manner. The generation of 4-POBN/radical adduct is dependent on the presence of NADPH, CPFX, and active microsomes. Furthermore, free radical production is completely inhibited by SKF 525A, Trolox, or ZnCl2.     

Hypochlorous acid as a precursor of free radicals in living systems

Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body’s inflammatory response.     

Role of free radicals on mechanism of radiation nephropathy

Purpose: In our study, after applying a single dose of 612 cGy irradiation, we aimed to observe the role of free radicals on tissue damage in the kidney caused by radiation by measuring NO level, Na/K-ATPase activity and TBARS amount which is an indicator of free radical damage. On the other hand we investigated whether the tissue damage can be prevented by vitamin A or not. Materials and methods: This study was performed on three groups: 1. Control group 2. The group to which irradiation was administrated 3. The group which was given radiation + vitamin A. The irradiation group of animals were given a single dose of gamma irradiation at a sublethal dose. In the group which was administrated both irradiation + vitamin A, vitamin A was given for two days prior to irradiation. The amount of NO was measured by ESR spectroscopy, Na/K-ATPase and TBARS were measured by spectrophotometry. Results and conclusions: As a result of radiation mediated tissue damage in the kidney, we observed a NO loss, a decrease in Na/K-ATPase activitiy and an increase in TBARS amount. Although the administration of vitamin A before radiation, did not have any effect on NO loss and decrease in Na/K-ATPase.     

Generation of oxygen free radicals during the metabolism of cyclosporine A: a cause-effect relationship with metabolism inhibition

A better understanding of the mechanism of lipid peroxidation during the metabolism of cyclosporine A (CsA) might help explain the toxicities of this immunosuppressive drug on various organs. Ourin vitro work used microsomes prepared from livers of phenobarbital-induced male rats. The incubations (total volume 1ml) also contained a NADPH regenerating system and substrate (i.e., CsA, carbon tetrachloride, or aminopyrine) dissolved in ethanol. Lipid peroxidation was inferred from the presence of malondialdehyde (MDA) which was detected by the thiobarbituric acid assay. The formation of CsA hydroxylated metabolites (AM9 and AM1) was monitored by liquid chromatography. The activity of the microsomal incubation was confirmed by measurements of MDA and formaldehyde production caused by increasing concentrations of CsA, carbon tetrachloride, and aminopyrine. The occurrence of hydroxylated metabolites was not coupled to the production of MDA. Aminopyrine could inhibit MDA production by CsA, but CsA could not reduce the formation of formaldehyde by aminopyrine. Erythromycin, a competitor for the binding site of CsA on cytochrome P450, reduced MDA production by CsA, and CsA inhibited formaldehyde production by erythromycin. Interaction studies with SKF 525A, ketoconazole, superoxide dismutase, catalase, -tocopherol, and reduced glutathione confirmed the role of cytochrome P450 and the presence of activated oxygen species as a source of microsomal peroxidation which in return may explain the inhibitory effect of CsA on cytochrome P450 itself.Abbreviations AM9 9hydroxycyclosporine - AM1 1(8)hydroxycyclosporine - AM1c 1hydroxy--cyclo-cyclosporine - AM4N 4N-desmethylcyclosporine     

Detection of free radicals as a consequence of rat intestinal cellular drug metabolism

Because the intestine is the first pass organ for orally administered drugs and because some of these drugs are known to undergo oxidative metabolism leading to the formation of free radicals, we investigated the potential for this to occur in cell suspensions of rat enterocytes. As part of our study, the effect of intracellularly produced superoxide on cellular metabolism was investigated. The drugs chosen were the quinone, menadione and the aromatic nitro-containing compound, nitrazepam. On incubation of both drugs with isolated enterocytes and the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), rapid appearance of an electron paramagnetic resonance (EPR) spectrum was recorded which was characteristic of hydroxyl radicals being spin trapped by DMPO giving 2,2-dimethyl-5-hydroxy-1-pyrrolidenyloxyl (DMPO-OH). Experiments were conducted which determined that the EPR spectrum of DMPO-OH resulted from the initial spin trapping of superoxide by DMPO to yield the corresponding nitroxide, 2,2-dimethyl-5-hydroxyl-1-pyrrolidenyloxyl (DMPO-OOH). Bioreduction of DMPO-OOH by glutathione peroxidase led to the rapid formation of DMPO-OH. We believe this enzymic pathway accounted for the EPR spectrum noted in incubations with either drug in the presence of the spin trap, DMPO. The incubation of enterocytes with both drugs did not mediate release of 51Cr nor lactate dehydrogenase. However, production of 14CO2 from [14C]glucose was severely inhibited (4-5-fold) in the presence of both drugs, while the incorporation of [14C]leucine into trichloroacetic acid precipitable protein was antagonized by menadione only. We conclude that superoxide can be demonstrated to arise as the result of enterocyte metabolism of menadione or nitrazepam. The consequence of oxidative metabolism of these drugs results in cellular dysfunction.     

Detection of free radicals during the cellular metabolism of adriamycin

Experiments were conducted to determine which free radicals are generated during the metabolism of adriamycin (ADM) by canine tracheal epithelial (CTE) cells, guinea pig enterocytes, and rat hepatocytes. The technique employed in this study was spin trapping; the spin trap utilized was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed during the metabolism of ADM by CTE cells. However, the addition of dimethyl sulfoxide to the in vitro system suggested that superoxide is initially spin trapped by the nitrone, and that the adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) is rapidly bioreduced to afford DMPO-OH. The addition of superoxide dismutase to the system indicated that superoxide generation was primarily intracellular. The adriamycin semiquinone free radical (ADM-SQ) was produced during the metabolism by enterocytes and hepatocytes. The rate of the production of ADM-SQ was enhanced under anaerobic conditions, suggesting that molecular oxygen was responsible for the degradation of this carbon-centered free radical. However, spin trapping of oxygen radicals was not observed; this observation suggests that these reactive intermediates are not produced at concentrations sufficient for detection by spin-trapping experiments.     

LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.