Orientador: Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T16:24:35Z (GMT). No. of bitstreams: 1 RinckJunior_JoseAugusto_D.pdf: 4925898 bytes, checksum: cf6e78a6e4ad84c7bbe48d34d75fdae6 (MD5) Previous issue date: 2015 / Resumo: As glutationa S-transferases (GSTs) são enzimas detoxificantes. Os genes GSTM1 e GSTT1 são polimórficos e quando deletados perdem a expressão enzimática. As proteínas codificadas pelos genes XPD são responsáveis pelo reparo de lesões do DNA causadas pela luz solar. Polimorfismos nestes genes também podem codificar proteínas com funções comprometidas, em especial o Lys751Gln e o Asp312Asn do gene XPD. Ainda não é claro o papel dos polimorfismos destes genes no risco de melanoma cutâneo (MC) ou se estão associados com os aspectos clínicopatológicos. Foram incluídos 489 indivíduos (231 pacientes, 258 controles). A genotipagem foi realizada por reação em cadeia da polimerase e digestão enzimática. O risco de MC esteve aumentado em 2,00 (IC 95%: 1,05-3,81, P= 0,03) vezes em portadores do genótipo GSTT1 nulo + Asp/Asn + Asn/Asn do XPD Asp312Asn. O GSTT1 nulo elevou o risco de MC metastático em 3,75 (IC 95%: 1,48-9,44, P= 0,006) vezes e se combinado ao GSTM1 nulo em 7,33 (IC 95%: 2,09-25,68, P= 0,003) vezes. O alelo 312Asn elevou o risco de MC no tronco ou membros em 1,80 (IC 95%: 1,19-2,73, P= 0,005) vezes e do subtipo extensivo superficial ou nodular em 1,80 (IC 95%: 1,14-2,84, P= 0,01) vezes. Os genótipos Asn/Asn + Gln/Gln elevou o risco de MC de níveis I, II ou III de Clark em 2,46 (IC 95%: 1,12-5,37, P= 0,02) vezes. Os genótipos GSTM1 nulo + GSTT1 nulo (HR: 3,18; IC95%: 1,21-8,36, P= 0,01) e o genótipo GSTT1nulo + Gln/Gln (HR: 5,93; IC95%: 1,53-22,91, P= 0,01) estiveram associados a maior risco de morte. Concluímos que os referidos polimorfismos em combinações específicas podem aumentar a susceptibilidade ao MC e influenciar suas características clínicopatológicas e sobrevida / Abstract: The glutathione S-transferases (GST) are detoxifying enzymes; the GSTM1 and GSTT1 genes are polymorphic and when deleted lose enzyme expression. The XPD proteins are responsible for DNA damage repair caused by sunlight. Polymorphisms (SNPs) in XPD genes may also result proteins with impaired function, in particular Lys751Gln and Asp312Asn. However It¿s not entirely clear whether the SNPs of these genes influence the risk of cutaneous melanoma (CM) or are associated with clinic pathological aspects of this disease. In the present study 489 individuals were included (231 patients and 258 controls). Genotyping was performed by polymerase chain reaction and enzyme digestion. The risk of MC was increased 2.00-fold (95% CI: 1.05-3.81, P= 0.03) in carriers of the GSTT1 null combined with Asp/Asn + Asn/Asn genotype of XPD Asp312Asn. The GSTT1 null genotype and GSTT1 null + GSTM1 null genotype increased the risk of metastatic MC by 3.75-fold (95% CI: 1.48-9.44, P= 0.006) and 7.33-fold (95% CI: 2.09-25.68, P= 0.003), respectively. The allele 312Asn increased the risk of MC in the trunk or limbs in 1.80-fold (95% CI: 1.19-2.73, P= 0.005) and superficial spreading or nodular subtype in 1.80-fold (95%: 1.14-2.84, P= 0.01). The combined Asn/Asn + Gln/Gln genotype raised the risk of MC in Clark¿s level I, II or III in 2.46-fold (95% CI: 1.12-5.37, P= 0.02). The genotype GSTM1null + GSTT1 null (HR: 3.18; 95% CI: 1.21-8.36, P= 0.01) and GSTT1 null + Gln/Gln (HR: 5.93; 95% CI: 1.53-22.91, P= 0.01) were predictive of lower overall survival. In conclusion, polymorphisms in specific genes combinations may increase susceptibility to MC and influence their clinic pathological features and survival / Doutorado / Clinica Medica / Doutor em Clínica Médica

Orientador: Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T18:37:49Z (GMT). No. of bitstreams: 1 Pincinato_EderdeCarvalho_D.pdf: 3229024 bytes, checksum: a92cc669d0c70abe36aab0ff261da986 (MD5) Previous issue date: 2015 / Resumo: A cisplatina (CDDP) associada à radioterapia (RT) é utilizada no tratamento de portadores de carcinoma de células escamosas de cabeça e pescoço (CCECP). A resposta ao tratamento bem como seus efeitos colaterais variam de indivíduo para indivíduo e tal fato pode ser explicado pela variabilidade genética no metabolismo da CDDP. O objetivo deste estudo foi o de verificar se habilidades herdadas na detoxificação da CDDP, mediadas pelas enzimas GSTM1, GSTT1 e GSTP1, alteram os efeitos terapêuticos, colaterais e a concentração de CDDP urinária em pacientes com CCECP. Foram avaliados, de forma prospectiva, 90 pacientes com CCECP tratados com CDDP associada à RT. Os genótipos dos polimorfismos GSTM1, GSTT1 e GSTP1 Ile105Val foram analisados por meio da reação em cadeia da polimerase (PCR) multiplex e PCR seguida de digestão enzimática, respectivamente, em DNA de sangue periférico. A resposta ao tratamento foi avaliada por exame clínico e tomografia computadorizada do pescoço. Os efeitos colaterais ao tratamento foram graduados por questionários e exames laboratoriais. As toxicidades renal e auditiva foram avaliadas por clearance de creatinina estimado (ClCrea), taxa de filtração glomerular com EDTA-51Cr (TFG) e audiometria tonal limiar. As concentrações urinárias da CDDP foram realizadas por cromatografia líquida de alta eficiência (HPLC). Pacientes com a deleção homozigótica do gene GSTT1 estiveram sob risco 0,09 (IC 95%: 0,02-0,41) vezes menor de ocorrência de vômitos, 0,22 (IC 95%: 0,05-0,89) vezes menor de ototoxicidade e menor redução de ClCrea (81,69 ± 21,40 para 84,13 ± 25,69 versus 93,16 ± 28,94 para 77,52 ± 23,96 mL/min/1,73m²) e de TFG (79,56 ± 20,68 para 69,94 ± 21,40 versus 84,38 ± 19,96 para 62,87 ± 20,72 mL/min/1,73m²) do que os portadores do gene. Já os pacientes com o alelo Val do polimorfismo GSTP1 Ile105Val estiveram sob risco 6,32 vezes maior de ocorrência de vômitos acentuados (IC 95%: 2,05-19,51), 3,35 vezes maior de ototoxicidade acentuada (IC 95%: 1,03-10,96) e maior redução de TFG (80,87 ± 21,73 para 66,97 ± 24,96 versus 85,35 ± 18,76 para 62,19 ± 17,40 mL/min/1,73m²) do que os portadores do genótipo Ile/Ile. Maior concentração urinária de CDDP foi observada em pacientes com a deleção homozigótica do gene GSTT1, quando comparados aos pacientes com a presença do gene (429,58 ± 116,24 versus 253,42 ± 95,20 ug CDDP/mg creatinina). Concluímos que estes polimorfismos desempenham papéis importantes na ocorrência de eventos adversos da terapêutica e excreção urinária do CDDP. Acreditamos que estes resultados possam constituir a base preliminar para o tratamento personalizado futuro de pacientes com CCECP / Abstract: Cisplatin (CDDP) associated with radiotherapy (RT) is used in treatment of patients with head and neck squamous cell carcinoma (HNSCC). The response to treatment as well as its side effects vary among individuals, and this fact may be explaned by the genetic variability in metabolism of CDDP. The aim of this study was to access if inherited ability to cellular CDDP detoxification, mediated by GSTM1, GSTT1 and GSTP1 enzymes alters the therapeutic and side effects of CDDP and RT and urinary concentration of CDDP in HNSCC patients. We evaluated, prospectively, 90 consecutive HNSCC patients, who received CDDP associated RT as treatment. Genotypes of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms were analyzed by multiplex polymerase chain reaction (PCR) and PCR followed by restriction enzyme digestion, respectively, in peripheral blood DNA. Treatment response was assessed by clinical examination and computed tomography of neck. Treatment side effects were ranked through questionnaire and laboratory tests. Renal and hearing toxicities were assessed using, respectively, estimated creatinine clearance and glomerular filtration 51Cr-EDTA and pure tone threshold audiometry. Urinary doses of CDDP were performed by high performance liquid chromatography (HPLC). Patients with the GSTT1-null genotype had a 0.09 times (95% CI:0.02-0.41) decreased risk for vomiting, 0.22 times (95% CI:0.05-0.89) decreased risk for ototoxicity, less creatinine clearance decreases (81.69 ± 21.40 to 84.13 ± 25.69 versus 93.16 ± 28.94 to 77.52 ± 23.96 mL/min/1.73m²) and glomerular filtration 51Cr-EDTA (79.56 ± 20.68 to 69.94 ± 21.40 versus 84.38 ± 19.96 to 62.87 ± 20.72 mL/min/1.73m²) than GSTT1-present genotype. Patients with Val allele of GSTP1 Ile105Val polymorphism had 6.32 higher risk presenting high grade vomiting (95% CI: 2.05-19.51), 3.35 higher risk to high grade ototoxicity (95% CI: 1.03-10.96) and higher glomerular filtration 51Cr-EDTA reduction (80.87 ± 21.73 to 66.97 ± 24.96 versus 85.35 ± 18.76 to 62.19 ± 17.40 mL/min/1.73m²) when compared to wild genotype. Higher CDDP urinary level was observed in patients with the GSTT1-null genotype, compared to patients with the gene (429.58 ± 116.24 versus 253.42 ± 95.20 ug CDDP/mg creatinine). We concluded that these genetic polymorphisms have important roles in complete response rate, in occurrence of side effects, and in urinary CDDP excretion. We believe that this data may constitute preliminary basis of future personalized treatment of HNSCC patients / Doutorado / Clinica Medica / Doutor em Clínica Médica

Les protéines destinées à la sécrétion ou adressées à la membrane transitent par le réticulum endoplasmique (RE) où elles acquièrent leur conformation native et subissent des modifications post-traductionnelles comme la formation de ponts disulfures. Dans ce compartiment, la formation de ponts disulfures repose sur l’activité de l’oxydase Ero1 et de la Protein Disulfure Isomerase (PDI). Ero1 catalyse la formation de ponts disulfures et les transmet à la PDI qui à son tour oxyde les substrats. L’isomérisation ou la réduction terminale des ponts disulfures non-natifs repose sur un système de réduction dans le RE encore non élucidé. Des études suggèrent l’importance du glutathion réduit (GSH) dans ce système de réduction. Le GSH est un tripeptide redox exclusivement synthétisé dans le cytosol. Notre étude s’attache à (i) décrire les flux de glutathion entre RE et cytosol et (ii) identifier les acteurs de ce transport (iii) comprendre l’impact d’une modification de l’homéostasie redox du glutathion sur la physiologie du RE.Nous avons établi un système permettant d’étudier les flux de glutathion entre cytosol et RE. Afin de démasquer ces flux intracellulaires, nous avons utilisé une souche de S. cerevisiae surexprimant le transporteur plasmatique du glutathion, HGT1. Ce système permet de modifier rapidement et drastiquement la concentration cytosolique de glutathion. Les flux intracellulaires engendrés sont ensuite suivis grâce à des sondes redox spécifiques du glutathion adressées dans le RE ou le cytoplasme.(i) Nos résultats suggèrent que le GSH et le GSSG sont importés dans le RE depuis le cytosol. Le GSH est transporté selon un gradient de concentration via un système de transport de diffusion facilité. Ces flux sont également observés lors de stress stimulant la synthèse de GSH (stress thermique, arsenite…).(ii) Le transport de GSH dans le lumen est assuré par le translocon Sec61, et une régulation de cet import par la chaperone luminale Kar2 est observée.(iii) une réduction rapide de l’état redox du glutathion dans le RE conduit à une mort cellulaire programmée non apoptotique, également observée lors d’autre stress RE (traitement tunicamycine). / The endoplasmic reticulum (ER) is the first intracellular compartment of the protein secretion pathway. Protein maturation in this compartment involves protein folding and post-traductionnal modification including formation of disulfide bonds. The formation of disulfide bonds is operated by a highly conserved redox relay made of the thiol oxidase Ero1 and the protein disulfide isomerase (PDI). Ero1p catalyzes disulfide bond formation and relays them by thiol-disulfide exchange to PDI, which in turn oxidizes substrates. Isomerization and terminal reduction of non-native disulfide bonds both rely on a reduction system that remains to be formally identified. Studies however suggest the importance of reduced glutathione in this reducing system. GSH is small redox tripeptide exclusively synthesized in the cytosol. In this study we (i) describe the main parameters of glutathione traffic across the ER membrane (ii) identify the main actors involved in the transport and (iii) analyze the physiological impact of a modification of the ER glutathione redox state.We established a system to monitor the fluxes of glutathione from the cytosol to the ER in S. cerevisiae. To artificially increase fluxes of glutathione, we used a cell over-expressing the GSH plasma membrane transporter HGT1, which when grown in presence of glutathione import high levels of this compound. Consequently, we monitored the intracellular relocation of imported GSH by following GSH fluxes using two specific redox probes. Our data indicate that:(i) GSH is transported into the ER by facilitated diffusion along a concentration gradient. GSSG can also be imported into the ER. Similarly, stress conditions that stimulate GSH synthesis, such as heat shoc, arsenite treatment, also triggered a GSH import in the ER.(ii) GSH import in the ER is achieved by the translocon Sec61, and is regulated by the lumenal chaperone Kar2.(iii) A rapid reduction of glutathione ER redox state leads to the activation of a non-apoptotic programmed cell death pathway, usually observed during high ER stress.

During the past few years, gold nanoparticles (AuNPs) have received considerable attention in many fields due to their optical properties, photothermal effect and biocompatibility. AuNPs, particularly AuNCs and AuNRs, exhibit great potential in diagnostics and imaging. In the present study, AuNCs were used to selectively image and quantify intracellular antioxidants. It was reported by Chen et al. that the strong fluorescence of AuNCs is quenched by highly reactive oxygen species (hROS). Most of applications depend on fluorescence quenching, however, for our project we designed turn-on fluorescent sensors using AuNCs that sense antioxidants. In the presence of antioxidants, AuNCs fluorescence switch on, while in the absence of antioxidants their fluorescence immediately turn off due to hROS effect. AuNRs were also used for cellular imaging in which AuNRs were conjugated to Cy3-labelled molecular beacon (MB) DNA. Next, the previous complex was loaded in two different strains of magnetotactic bacteria (MTB). MTB were used as a targeted delivery vehicle in which magnetosomes direct the movement of bacteria. The DNA sequence was specific to a certain sequence in mitochondria. The exposure of MTB to an alternating magnetic field (AMF) leads to the increase of temperature inside the bacteria, which destruct the cell wall, and hence, bacterial payload is released. When MD-DNA hybrid with the target sequence, AuNR and Cy3 separate from each other, the fluorescence of the Cy3 is restored.

UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.

Cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species and a decreased expression of enzymatic antioxidants. Prodrugs inducing additional oxidative stress can selectively induce apoptosis in cancer cells already having a high level of intrinsic oxidative stress. This study focused on the rational design and in vitro evaluation of a novel prodrug ester, (4- [(nitrooxy)methyl]phenyl-N-acetyl-L-alaninate or NPAA) activated by acylamino-acidreleasing enzyme (AARE, EC 3.4.19.1) to yield a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. NPAA shares structural features with both nitric oxide donating aspirin (NO-ASA), a wellcharacterized QM releasing anticancer prodrug, and N-acetyl-L-alanine-4-nitroanailide (AANA), a known specific substrate for AARE. AARE is a serine peptidase that is overexpressed in some tumors and cancer cell lines. The overall approach was to first predict the 3-dimensional structure of both rat (rAARE) and human AARE (hAARE) and then use the resulting low-resolution models to determine if NPAA was a plausible prodrug by estimating its affinity to hAARE and rAARE in comparison to AANA. The AARE models were constructed using a bioinformatic-based protein structure prediction webserver (I-TASSER) followed by energy minimization and refinement. The resulting models were subjected to a variety of structural quality assessments. The optimal models of hAARE and rAARE were found to have similar three-dimensional structures with a ß- propeller domain and an a/ß-hydrolase domain containing an exopeptidase catalytic site with active site residue distances typically found in serine peptidases. Protein-ligand docking studies showed that both AANA and NPAA could bind to the exopeptidase catalytic site of the hAARE and rAARE models with reasonable affinities and in a region with a highly druggable pocket. In order to validate the in silico results, NPAA was synthesized, purified, physically characterized and evaluated for its in vitro ability to deplete GSH in the presence of rAARE. As anticipated, NPAA was found to deplete GSH and this effect was completely blocked by diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, including rAARE. These studies support further efforts to optimize the design of QM releasing anticancer prodrugs targeted to AARE. Moreover, the molecular models presented here could be useful for the rational design of AARE inhibitors, which could also be exploited as potential anticancer agents.

Glutamate, an excitatory neurotransmitter in the central nervous system, plays an important role in neurological disorders. Previous studies have shown that excess glutamate can cause oxidative stress in a hippocampal HT-22 cell line. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as stroke and Parkinson's disease. In this study, we found that there is no TrkB receptor in HT-22 cells. Despite this, our data demonstrate that 7,8-DHF still protects against glutamate-induced toxicity in HT-22 cells in a concentration-dependent manner, indicating that 7,8-DHF prevents cell death through other mechanisms rather than TrkB receptors in this cell model. We further show that 7,8-DHF increases cellular glutathione levels and reduces reactive oxygen species (ROS) production caused by glutamate in HT-22 cells. Finally, our data demonstrate that 7,8-DHF protects against hydrogen peroxide and menadione-induced cell death, suggesting that 7,8-DHF has an antioxidant effect. In summary, although 7,8-DHF is considered as a selective TrkB agonist, our results demonstrate that 7,8-DHF can still confer neuroprotection against glutamate-induced toxicity in HT-22 cells via its antioxidant activity.

Free-radical generation is one of the primary causes of myocardial ischemia/reperfusion (I/R) injury. Melatonin is an efficient free-radical scavenger and induces the expression of antioxidant enzymes. We have previously shown that melatonin can prevent free-radical-induced myocardial injury. To date, the mechanism underlying melatonin's cardioprotective effect is not clear. In this study, we assessed the ability of melatonin to protect against I/R injury in mice deficient in glutathione peroxidase 1 (Gpx1). Mice hearts were subjected to 40 min of global ischemia in vitro followed by 45 min of reperfusion. Myocardial I/R injury (expressed as % of recovery of left ventricular developed pressure × heart rate) was exacerbated in mice deficient in Gpx1 (51 ± 3% for Gpx1+/+ mice versus 31 ± 6% for Gpx1-/- mice, P < 0.05). Administration of melatonin for 30 min protected against I/R injury in both Gpx1+/+ mice (72 ± 4.8%) and Gpx1-/- mice (63 ± 4.7%). This protection was accompanied by a significant improvement in left ventricular end-diastolic pressure and a twofold decrease in lactate dehydrogenase (LDH) level released from melatonin-treated hearts. In another set of experiments, mice were subjected to 50 min of ligation of the left descending anterior coronary artery in vivo followed by 4 hr of reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly larger in Gpx1-/- mice than in Gpx1+/+ mice (75 ± 9% versus 54 ± 6%, P < 0.05) and were reduced significantly in melatonin-treated mice (31 ± 3.7% Gpx1-/- mice and 33 ± 6.0% Gpx1+/+ mice). In hearts subjected to 30 min of coronary artery occlusion followed by 3 hr of reperfusion, melatonin-treated hearts had significantly fewer in situ oligo ligation-positive myocytes and less protein nitration. Our results demonstrate that the cardioprotective function of melatonin is independent of Gpx1.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H2O2, which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H2O2. In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H2O2 generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.