Eduard Berenshtein

Plasma antioxidant status and cell injury after severe physical exercise

Strenuous exercise leads to an increase in metabolic rate, increased production of reactive oxygen species, and compromised antioxidant defense systems. To study the effects of oxidative stress during strenuous exercise, a homogeneous group of 31 male subjects participated in a 6-month, 5 days/week training schedule involving two extreme marches of 50 km and 80 km at sea level, separated by 2 weeks of regular training. Each participant carried 35 kg of extra weight. Blood samples were drawn imediately before and after each march. Twenty-nine subjects completed the 50-km march, and only 16 completed the 80-km march. Plasma levels of reduced ascorbic acid, total ascorbate, and dehydroascorbate did not undergo significant changes during either march. However, the 50- and 80-km marches led to 25% and 37% increases, respectively, in plasma levels of uric acid; due presumably to increases in the metabolic rate and consequent pyrimidine nucleotide metabolism. Both marches led to ≈10-fold increase leakage of creatine phosphokinase into the plasma. Likewise, plasma levels of aspartate transaminase, a characteristic marker of liver injury, increased ≈4-fold. Plasma levels of bilirubin, creatine, urea, and glucose also increased. Plasma protein carbonyl content, a marker of protein oxidative damage, decreased significantly during each march. These results are discussed with respect to the consideration that elevation of the respiration rate during exercise leads to production of more reactive oxygen species than the antioxidant systems can scavenge. Plausible explanations for leakage of molecules into the plasma are discussed.

The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent

The opportunistic pathogen Pseudomonas aeruginosa causes infections that are difficult to treat by antibiotic therapy. This bacterium can cause biofilm infections where it shows tolerance to antibiotics. Here we report the novel use of a metallo-complex, desferrioxamine-gallium (DFO-Ga) that targets P. aeruginosa iron metabolism. This complex kills free-living bacteria and blocks biofilm formation. A combination of DFO-Ga and the anti-Pseudomonas antibiotic gentamicin caused massive killing of P. aeruginosa cells in mature biofilms. In a P. aeruginosa rabbit corneal infection, topical administration of DFO-Ga together with gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of gentamicin alone. The use of DFO-Ga as a Trojan horse delivery system that interferes with iron metabolism shows promise as a treatment for P. aeruginosa infections.

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron — providing protection to the ischemic heart via preconditioning (PC).

HU-331, a novel cannabinoid-based anticancer topoisomerase II inhibitor

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331–caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug. [Mol Cancer Ther 2007;6(1):173–83]

Prophylactic administration of topical glutamine enhances the capability of the rat colon to resist inflammatory damage

Glutamine is an important nutrient for the GI tract and has been shown to exert a protective effect on the bowel. Nonetheless, in the context of IBD, data demonstrating a therapeutic role for glutamine has been inconclusive. IBD is associated with oxidative stress caused by reactive oxygen species. We aimed to investigate the effect of topical glutamine administration in rats before or after induction of colitis by trinitrobenzenosulfonic acid. In study I glutamine enemas were given beginning 2 days before or on the same day of induction of colitis. Inflammation severity was assessed by macroscopic and microscopic score and tissue myeloperoxidase activity. In study II glutamine enemas were given for 3 days without induction of colitis: mitotic index and colonic crypt length were measured, as well as water-soluble low molecular weight antioxidants and energy-rich phosphate levels (by HPLC). Results showed that glutamine significantly decreased indexes of inflammation when administered before induction of colitis. Glutamine caused an increase in the mitotic index and the levels of water-soluble low molecular weight antioxidants and energy-rich phosphates. We conclude that glutamine exerts a beneficial effect only when administered before induction of colitis, by increasing the resistance of the colonic tissue to inflammatory injury. This effect is probably mediated by increasing the antioxidant capacity and energy level of the tissue.

Nitroxide Radicals Prevent Metal-aggravated Reperfusion Injury in Isolated Rat Heart

The effects of Cu(II) and the stable nitroxide radical 4-OH-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TPL) on reperfusion injury following global myocardial ischemia have been studied using the isolated rat heart model in the Langendorff configuration. Hearts were equilibrated with Krebs-Henseleit buffer (KH-buffer) for 10 min and subjected to 18 min of normothermic global ischemia. After 20 min reperfusion, hemodynamic parameters recovered as follows: ventricular developed pressure (77%), dP/dt (71%) and -dP/dt (80%), heart rate (91%), and work index (70%). End-diastolic pressure was 16 mm Hg. When 10 microM Cu-nitrilotriacetate or Cu-(histidine)2 was included in the perfusate before, during, and following ischemia, the heart injury was more extensive and the work index only recovered to 17% of the preischemic value. The inclusion of 100 microM TPL during reperfusion abolished the copper-induced sensitization. In the absence of copper, TPL did not provide any protection against ischemia-reperfusion damage to the heart. The inclusion of 100 microM 1,4-dihydroxy-2,2,6,6-tetramethylpiperidine (TPL-H) during reperfusion, partially abolished the copper-induced sensitization. Since conversion between TPL and TPL-H takes place, the fact that both forms provide protection can increase their protective efficacy.

Cardiac Protection by Preconditioning Is Generated via an Iron-Signal Created by Proteasomal Degradation of Iron Proteins

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Rat Cardiac Mitochondrial Sub-populations Show Distinct Features of Oxidative Phosphorylation during Ischemia, Reperfusion and Ischemic Preconditioning

Background: Inter-fibrillar (IFM) and sub-sarcolemmal (SSM) mitochondria are two distinct mitochondrial sub-populations and are expected to behave differently during pathological conditions. This study was undertaken to compare functional oxidative phosphorylation (OXPHOS) in IFM and SSM during ischemia, reperfusion and ischemic preconditioning. Methods: Langendorff perfused Wistar rat hearts were subjected to 35minutes ischemia, 60minutes reperfusion and ischemic preconditioning (IPC) procedure (3cycles of 2-minutes ischemia followed by 3-minutes reperfusion).Subsequently IFM and SSM were isolated, and mitochondrial electron transport chain (ETC) enzyme activities and respiration were measured immediately. Results: Functional enzyme activities of ETC in IFM and SSM showed prominent differences especially in the proximal part of ETC enzymes during ischemia and reperfusion. SSM favor FADH2 while IFM prefer NADH as the main reducing equivalent for electron transport during ischemia and reperfusion. IPC preserved ETC enzyme activities in both IFM and SSM rendering cardio protection. Similarly IPC preserve ADP stimulated respiration with glutamate and malate as substrate in both sub populations, but not in IFM, with succinate as substrate. Apparently, the preconditioning imparts enhanced protection more to SSM than IFM during ischemia and reperfusion and especially to the proximal part of the ETC. Conclusion: We propose that mitochondrial dysfunction, one of the major targets of myocardial ischemia reperfusion injury needs to be evaluated by the synergic effect of both IFM and SSM.

The Effect of Restraint Stress on the Normal Colon and on Intestinal Inflammation in a Model of Experimental Colitis

Stress may induce development of inflammation in animal models of colitis. The effects of restraint stress on oxidative damage and on antioxidants in the normal colonic mucosa were studied. The effect of stress on the severity of indicators of inflammation, as well as the importance of mucosal substance P (SP) as a mediator of this effect were investigated in the TNBS-colitis model. Restraint stress significantly increased malondialdehyde levels and reduced levels of low-molecular-weight-antioxidants in the normal colon. ATP and the mucosal “energy charge” decreased substantially with chronic stress. Chronic stress worsened the extent of inflammation in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Mucosal SP content was not affected by exposure to chronic stress but increased after induction of colitis. The increase was greater when colitis was induced after exposure to stress. We conclude that chronic restraint stress causes oxidative damage to the normal colon and aggravates intestinal inflammation induced by TNBS. This effect may be mediated by SP.

Flumazenil mimics whereas midazolam abolishes ischemic preconditioning in a rabbit heart model of ischemia-reperfusion.

Background:The goal of the current study was to assess the effects of flumazenil, a benzodiazepine receptor antagonist, in limiting infarct size and in reducing hydroxyl free radical production. Methods:After intravenous salicylate (100 mg/kg) administration, rabbits were subjected to 40 min of regional myocardial ischemia and 2 h of reperfusion. In one group, flumazenil (0.05 mg/kg) and, in another, midazolam (0.05 mg/kg) was administered 15 min before 40 min of ischemia. Ischemic preconditioning (IP) was elicited by 5 min of ischemia followed by 10 min of reperfusion (before the 40-min ischemia period). In two other groups, midazolam was added to flumazenil and IP. Infarct size was determined using triphenyl tetrazolium chloride staining. The authors quantified the hydroxyl-mediated conversion of salicylate to its 2,3- and 2,5-dihydroxybenzoate derivatives during reperfusion by high-performance liquid chromatography coupled with electrochemical detection. Results are expressed as mean ± SEM. Results:Flumazenil, like IP, significantly decreased infarct size (23 ± 4 and 22 ± 5%, respectively, vs. 57 ± 6% in control group; P < 0.01). Midazolam inhibited the effects of flumazenil and IP. Flumazenil and IP significantly limited the increase in the normalized concentrations of 2,3- and 2,5-dihydroxybenzoic acids. With midazolam, however, the increase was comparable to that of the control group. 5-Hydroxydecanoate, a selective mitochondrial adenosine triphosphate–sensitive K+ channel blocker, given with flumazenil, abolished the protection obtained with the latter. Conclusions:Flumazenil mimics preconditioning to decrease infarct size and hydroxyl radical production during reperfusion. Midazolam, however, abolishes these effects. Blockade of benzodiazepine receptors is upstream to the mitochondrial adenosine triphosphate–sensitive K+ channels in the preconditioning cascade.