Maria C. Jordani

Flufenamic acid as an inducer of mitochondrial permeability transition.

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3′-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (ΔΨ), osmotic swelling, Ca2+ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 μM Ca2+, 5 μM ruthenium red (RR) or 1 μM cyclosporin A (CsA) were used. The dose response-curves for both respiration release and ΔΨ dissipation were nearly linear, presenting an IC50 of approximately 10 μM and reaching saturation within 25-50 μM, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 μM Ca2+, followed by ΔΨ dissipation and Ca2+ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca2+. ADP, Mg2+, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H2O2 production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.

Should Preconditioning Hyperbaric Oxygenation Protect the Liver Against Ischemia–Reperfusion Injury? An Experimental Study in a Rat Model

OBJECTIVES We designed studies to test the hypotheses that hyperbaric oxygen (HBO) therapy should protect liver against subsequent ischemia/reperfusion (I/R) injury are scarce and controversial. The purpose of this study was to clarify some questions about the association of HBO with the processes of liver I/R. METHODS We divided Wistar rats into 5 groups: (1) SHAM operation, (2) I/R, rats submitted to total pedicle ischemia for 30 minutes followed by 5 minutes of reperfusion; (3) HBO60I/R and (4) HBO120I/R, rats respectively submitted to 60 and 120 minutes of HBO therapy at 2 absolute atmospheres and immediately after submitted to the experimental protocol of I/R; (5) HBO120, rats submitted to 120 minutes of HBO therapy at 2 absolute atmospheres and then immediately after humanely killed. The experimental protocol included (1) serum levels of aspartate and alanine aminotransferase; (2) mitochondrial function; (3) tissue malondialdehyde (MDA); and (4) plasma nitrite/nitrate. Data were analyzed using the Mann-Whitney test and were considered significant P < 5%. RESULTS The processes of liver ischemia/reperfusion caused tissue injury with hepatic mitochondrial functional impairment. A single exposure to 120 minutes of HBO caused an increase of tissue MDA. The time of HBO exposure as preconditioning before hepatic I/R is critical in the prevalence of beneficial or deleterious effects. Sixty minutes of hyperoxic preconditioning before liver I/R presents systemic benefits, but no significant tissue preservation. One hundred twenty minutes of hyperoxic preconditioning tissue liver benefits predominate compared with systemic benefits. CONCLUSIONS The HBO preconditioning therapeutic benefits to liver I/R injury are time dependent, suggesting a therapeutic window that needs to be clearly defined in future studies.

Hyperbaric oxygen therapy and ischemia and reperfusion: a valuable association to attenuate ischemic lesion and hepatic reperfusion

PURPOSE To investigate the consequences of the association between hyperbaric oxygen therapy and hepatic ischemia / reperfusion. METHODS Wistar rats were divided into three groups: SHAM, rats submitted to surgical stress and anesthetic but not hepatic ischemia or reperfusion, I / R, rats submitted to total hepatic pedicle ischemia for 30 min, followed by 5 min of reperfusion; HBO120, rats submitted to 120 min of hyperbaric oxygen therapy at two absolute atmospheres and immediately after submitted to the experimental protocol of ischemia and reperfusion. The preservation of the hepatic function was evaluated by determining mitochondrial swelling and malondialdehyde tissue level, as well as alanine aminotransferase and aspartate aminotranferase serum levels. The results were analyzed using the Mann-Whitney test and differences were considered significant for p<0.05. RESULTS There were significant differences in values: mitochondrial swelling of the I / R group compared to SHAM and HBO120; malondialdehyde between SHAM vs. I / R, SHAM vs HBO120, and I / R vs HBO120, alanine aminotransferase between SHAM vs. I / R . There was no significant difference between groups in aspartate aminotransferase serum levels. CONCLUSION The association between hyperbaric oxygen therapy and hepatic ischemia and reperfusion process was positive.

Avaliação do intumescimento osmótico mitocondrial do fígado submetido a isquemia parcial e ao pré-condicionamento isquêmico

O objetivo deste trabalho foi avaliar a permeabilidade da membrana mitocondrial em animais submetidos a pre-condicionamento isquemico, isquemia e reperfusao seguido de hepatectomia a 70%. Foram utilizados 30 ratos Wistar divididos em 3 grupos de 10: Grupo Controle (fc), Grupo Isquemico (fi) e Grupo Pre-condicionado (fp). Apos o procedimento cirurgico os animais foram sacrificados para a remocao do remanescente hepatico (30%) do qual foram isoladas as mitocondrias para a realizacao dos estudos de permeabilidade da membrana mitocondrial interna atraves do intumescimento osmotico. Foi observado um aumento significativo do intumescimento osmotico mitocondrial do grupo fi em relacao aos grupos fc e fp, sugerindo uma possivel participacao de especies reativas do oxigenio no processo.

Função mitocondrial hepática compensatória na coletase extra hepática aguda

A atividade compensatoria mitocondrial e um mecanismo que permite a alguns orgaos manter seu nivel energetico em situacoes adversas. Este presente estudo visa analisar a funcao mitocondrial hepatica em ratos submetidos a OBEH. Foram estudados 10 ratos Wistar machos (180 a 230 g), divididos em dois grupos. Grupo operacao simulada - OS e grupo obstrucao biliar extra hepatica (OBEH). Apos 24 h. foram coletadas amostras de sangue e biopsia hepatica para a dosagem do nivel serico das enzimas hepaticas e a atividade mitocondrial. Houve aumento significativo das aminotranferases, da FA e BT (tabela 1).Verificou-se aumento do Estado 3 da respiracao mitocondrial. Analisando os resultados, observou-se que as mitocondrias hepaticas apresentaram atividade compensatoria no sentido de preservacao da funcao hepatocelular

Liver Mitochondrial Function in Familial Amyloidotic Polyneuropathy

The objective of the present study was to analyze hepatic mitochondrial function in patients with familial amyloidotic polyneuropathy (FAP) undergoing cadaveric donor orthotopic liver transplantation. From February 2005 to May 2007, eight patients with FAP, ranging in age from 34 to 41 years and with Model for End-Stage Liver Disease scores ranging from 24 to 29. Underwent orthotopic transplantation using a liver from a deceased donor by the piggyback method. Immediately before beginning the recipient hepatectomy in a patient with FAP, a biopsy was obtained for analysis of mitochondrial function (FAP group). The control group consisted of 15 patients undergoing hepatic surgery to treat small tumors of the liver. Mitochondrial respiration was determined on the basis of oxygen consumption by energized mitochondria using a polarographic method. The membrane potential of the mitochondria was determined spectrofluorometrically. Data were analyzed statistically by the Mann-Whitney test, with the level of significance set at 5%. State 3 and 4 values, respiratory control ratio, and membrane potential were 47 +/- 8 versus 28 +/- 10 natoms O/min/mg protein (P < .05); 14 +/- 3 vs 17 +/- 7 nat.O/min/mg.prot.mit. (P > .05); 3.6 +/- .5 vs 1.7 +/- 0.7 (P < .05); and 135 +/- 5.2 vs 135 +/- 6 mV (P > .05) for control versus FAP patients, respectively, demonstrating a decreased energy status of the liver in FAP.

Behavior of cholinesterase and liver mitochondrial function in dogs submitted to normothermic ischemia and reperfusion

PURPOSE: The plasmatic activity of the cholinesterase (CHE) and the liver mitochondrial function, expressed by the ratio of respiratory control (RCR), were studied during normothermic ischemia. METHODS: Sixteen adult mongrels, eight females and eight males were submitted to ischemia by clamping of the hepatic artery, portal vein and infrahepatic inferior vena cava, infra-hepatic, for two h, follwed by reperfusion for 1 h. The CHE and the mitochondrial function were evaluated at 60 and 120 min. of ischemia and at 15 and 60 minutes of reperfusion. RESULTS: The CHE decreased, significantly, during ischemia and in reperfusion. The RCR was decreased at 120 min. of ischemia, returning to the initial values on reperfusion. CONCLUSION: In this study, the CHE was a sensitive indicator of ischemic injury , suggesting irreversibility of ischemia injury. The RCR, by other side, showed a greater sensibility than the CHE in detection sense, during the studied period, the reversibility of the hepatic ischemic injury.