José Ignacio Ruiz-Sanz

tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals

tert-Butyl hydroperoxide (TBHP) mobilizes arachidonic acid (AA) from membrane phospholipids in rat hepatocytes under cytotoxic conditions, thus leading to an increase in intracellular AA, which precedes cell death. In the present work, the involvement of lipid peroxidation, thiol status, and reactive oxygen species (ROS) in the intracellular AA accumulation induced by 0.5 mM TBHP was studied in rat hepatocytes. Cells treated with TBHP maintained viability and energy status at 10 min. However, TBHP depleted GSH, as well as inducing lipid peroxidation and ROS formation, detected by dichlorofluorescein (DCF) fluorescence. TBHP also significantly increased (32.5%) the intracellular [14C]-AA from [14C]-AA-labelled hepatocytes. The phospholipase A(2) (PLA(2)) inhibitor, mepacrine, completely inhibited the [14C]-AA response. The addition of antioxidants to the cell suspensions affected the TBHP-induced lipid response differently. The [14C]-AA accumulation correlated directly with ROS and negatively with endogenous GSH. No correlation between [14C]-AA and lipid peroxidation was found. Promethazine prevented lipid peroxidation and did not affect the [14C]-AA increase. We conclude that TBHP stimulates the release of [14C]-AA from membrane phospholipids through a PLA(2)-mediated mechanism. Endogenous GSH and ROS play a major role in this effect, while lipid peroxidation-related events are unlikely to be involved. Results suggest that specific ROS generated in iron-dependent reactions, different from lipid peroxyl radicals, are involved in PLA(2) activation, this process being important in TBHP-induced hepatocyte injury.

Supplementation with docosahexaenoic acid in the last trimester of pregnancy: maternal-fetal biochemical findings.

The nutritional significance of long-chain polyunsaturated fatty acids (LCPS) during the perinatal period is becoming increasingly important. There are currently very few studies on dietary intervention during gestation. The aim of the study was to analyze the effect of docosahexaenoic acid (DHA) supplementation during pregnancy on levels in both the newborn and the mother. A randomized placebo controlled study was carried out on 20 pregnant women in study group receiving 200 mg/day of docosahexaenoic acid-(DHA) during the last trimester of pregnancy. Results in both groups (A supplemented, B non-supplemented) highlighted a decrease in plasma arachidonic acid (5.99 +/- 0.91 vs. 4.51 +/- 0.71 p<0.001 for group A and 5.84 +/- 0.71 vs. 4.80 +/- 0.51 p<0.01 for group B) in the baseline-final intra-group comparison. The intergroup comparison revealed a significant difference in plasma DHA at delivery: it was found to be higher in the population of supplemented pregnant women (3.17 +/- 0.26 vs. 2.77 +/- 0.31). The neonate population displayed no significant differences between the two groups. The results show that LCPS are consumed during the final stages of pregnancy and that oral supplementation with 200 mg/day of DHA is reflected in an increase in the plasma level of this fatty acid in the mother. One could speculate that there would be a corresponding increase in DHA bioavailability for the fetus.

Superoxide Anions Are Involved in Doxorubicin‐Induced ERK Activation in Hepatocyte Cultures

Abstract:  Doxorubicin (DOX), an antineoplastic agent widely used for the treatment of cancer, belongs to the anthracycline family of antitumor antibiotics. DOX may undergo one‐electron reduction to the corresponding semiquinone free radical by flavin‐containing reductases. Under aerobic conditions, the semiquinone radical reacts rapidly with oxygen to generate superoxide anion, undergoing redox cycling. At moderate concentrations, reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. We have shown that DOX increased phosphorylation of enzymes comprising mitogen‐activated protein (MAP) kinase cascades in primary hepatocyte cultures, and that this action was independent of oxidant damage. In particular, extracellular signal‐regulated kinase (ERK) was phosphorylated by the drug treatment. In this work, we have determined the possible involvement of particular free radicals in DOX‐induced ERK phosphorylation in hepatocyte cultures by using specific free radical scavengers. The levels of ERK phosphorylation were measured by Western blot analysis with an anti‐Thr202/Tyr204‐phosphorylated p44/p42 MAPK antibody. Deferoxamine (DFO; iron chelator), catalase (hydrogen peroxide‐removing enzyme), or α‐tocopherol (peroxyl‐radical scavenger) did not affect DOX‐increased ERK phosphorylation levels. However, the cell‐permeable superoxide dismutase mimetic MnTBAP and the flavin‐containing enzyme inhibitor diphenyleneiodonium reverted DOX‐induced effects. These results suggest that superoxide anions, probably generated by DOX metabolism, are involved in the effects of the anthracycline on the MAP kinase cascade activation.

Serum oxidizability and antioxidant status in patients undergoing in vitro fertilization

OBJECTIVE To evaluate the serum oxidizability and antioxidant status in women undergoing an in vitro fertilization (IVF) cycle and to assess the possible relationship of the oxidizability indexes with the pregnancy rate. DESIGN Prospective, longitudinal study. SETTING Public university and public university hospital. PATIENT(S) Systematically recruited cohort of 125 women undergoing either IVF or intracytoplasmic sperm injection (ICSI). INTERVENTION(S) Serum samples were collected before the beginning of the use of gonadotropins (basal) and the day of human chorionic gonadotropin (hCG) administration (final) during an IVF cycle. MAIN OUTCOME MEASURE(S) The Cu2+-induced serum oxidation in terms of the oxidation rate in the lag (Vlag) and propagation (Vmax) phases and the time at which the oxidation rate is maximal (tmax), and measurements of serum total antioxidant activity (TAA), tocopherol, hydrophilic antioxidants, malondialdehyde, and nitric oxide. RESULT(S) Albumin, urate, bilirubin, alpha-tocopherol and gamma-tocopherol, TAA, and tmax statistically significantly decreased after the IVF cycle. Conception cycles were associated with a serum more prone to oxidation compared with nonconception cycles. In multivariate logistic regression analysis, the difference (final-basal) of the oxidation index Vlag (OR 1.394) and the body mass index (OR 0.785) were independent predictors of pregnancy. CONCLUSION(S) Treatment with IVF induces the production of reactive oxygen species (ROS), which is reflected in a serum less protected against oxidation. The results also suggest a role for ROS in the occurrence of conception in IVF.

Lipid Oxidation Inhibitory Effects and Phenolic Composition of Aqueous Extracts from Medicinal Plants of Colombian Amazonia

Diverse plants of ethnobotanic interest in Amazonia are commonly used in traditional medicine. We determined the antioxidant potential against lipid peroxidation, the antimicrobial activity, and the polyphenol composition of several Amazonian plants (Brownea rosademonte, Piper glandulosissimum, Piper krukoffii, Piper putumayoense, Solanum grandiflorum, and Vismia baccifera). Extracts from the plant leaf, bark, and stem were prepared as aqueous infusions, as used in folk medicine, and added to rat liver microsomes exposed to iron. The polyphenolic composition was detected by reverse-phase HPLC coupled to diode-array detector and MS/MS analysis. The antimicrobial activity was tested by the spot-on-a-lawn method against several indicator microorganisms. All the extracts inhibited lipid oxidation, except the P. glandulosissimum stem. The plant extracts exhibiting high antioxidant potential (V. baccifera and B. rosademonte) contained high levels of flavanols (particularly, catechin and epicatechin). By contrast, S. grandiflorum leaf, which exhibited very low antioxidant activity, was rich in hydroxycinnamic acids. None of the extracts showed antimicrobial activity. This study demonstrates for the first time the presence of bioactive polyphenolic compounds in several Amazonian plants, and highlights the importance of flavanols as major phenolic contributors to antioxidant activity.

Ala16Val SOD2 polymorphism is associated with higher pregnancy rates in in vitro fertilization cycles

OBJECTIVE To investigate whether the Ala16Val polymorphism in the SOD2 gene, encoding for mitochondrial manganese superoxide dismutase (SOD), is associated with [1] infertility and [2] the pregnancy rate (PR) in IVF cycles. DESIGN Prospective case-control study. SETTING Public university and public university hospital. PATIENT(S) A total of 362 newborns (nonselected population) and 148 infertile women undergoing an IVF cycle, from which 44 became pregnant and 104 did not. INTERVENTION(S) Blood samples extracted from the patients and newborn umbilical cord. MAIN OUTCOME MEASURE(S) Genotype and allele distribution of the Ala16Val polymorphism in the SOD2 gene using the tetra-primer amplification refractory mutation system-polymerase chain reaction (PCR). RESULT(S) The polymorphism distribution of the subfertile women was similar to that of a nonselected population. The SOD2 Ala allele frequency was 49% both in controls and IVF patients. In IVF population the Ala/Ala SOD2 genotype was 25%, with a 28% Val/Val homozygous. In contrast, the Ala/Ala genotype was associated with higher PRs in IVF (47% in Ala/Ala vs. 23% in no Ala/Ala). A multivariate logistic regression analysis revealed that the Ala/Ala genotype was an independent predictor of pregnancy (odds ratio [OR] = 3.29), followed by the number of transferred embryos (OR = 2.37) and age (OR = 0.84). CONCLUSION(S) The Ala/Ala SOD2 genotype is a significant independent predictor of the occurrence of pregnancy in IVF. Data also support a role for antioxidant defense, particularly in the mitochondria, in conception in IVF.

Doxorubicin-induced MAPK activation in hepatocyte cultures is independent of oxidant damage

Abstract:  Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited on account of its toxicity. DOX cytotoxic effects have been associated with reactive oxygen species (ROS) generated during drug metabolism. ROS induce signaling cascades leading to changes in the phosphorylation status of target proteins, which are keys for cell survival or apoptosis. The mitogen‐activated protein kinase (MAPK) cascades are routes activated in response to oxidative stress. In this work, the effects of DOX on cytotoxicity, indicators of oxidative stress (malondialdehyde ‐MDA‐ and GSH), and the phosphorylation status of extracellular signal‐regulated kinases (ERKs), c‐Jun N‐terminal kinases (JNKs), and p38 kinases were analyzed in primary cultures of rat hepatocytes. DOX (1–50 μM) did not modify lactate dehydrogenase (LDH ) release into the medium, the levels of MDA (determined by high‐performance liquid chromatography [HPLC]) or the intracellular GSH during the incubation time up to 6 h. GSH levels from mitochondria extracted by Percoll gradient from cultured hepatocytes were not modified by DOX, thus excluding its depletion or any impaired mitochondrial uptake. Characterization of proteins by Western blot analysis revealed that DOX increased phosphorylation of p38 kinases and JNK1 and JNK2 in a dose‐ and time‐dependent manner. DOX also increased ERK2 phosphorylation at latter time points. In conclusion, DOX triggers activation of ERK, JNK, and p38 kinases in primary cultures of rat hepatocytes independently of oxidant damage.

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 microM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM-100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.

Glutathione peroxidase activity in seminal plasma and its relationship to classical sperm parameters and in vitro fertilization-intracytoplasmic sperm injection outcome

OBJECTIVE To relate the glutathione peroxidase (GPX) activity level in human seminal plasma with standard semen parameters and spermatozoa fertilization potential in terms of fertilization and pregnancy rates in an IVF program. DESIGN Prospective study. SETTING Human Reproduction Unit at Cruces Hospital (Vizcaya, Spain). PATIENT(S) Three hundred consecutive males from infertile couples participating in the IVF program. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Analysis of GPX activity in seminal plasma by spectrophotometry. RESULT(S) GPX activity in seminal plasma was significantly lower in patients with abnormal sperm as assessed by 1999 and 2010 World Health Organization (WHO) criteria, compared with normozoospermic individuals. There was a more significant decrease in those samples with severe sperm pathologies. GPX values were significantly lower in samples with severe asthenozoospermia, oligozoospermia, and teratozoospermia compared with normal samples. However, there was no correlation between GPX activity in seminal plasma in IVF patients and fertilization rates or pregnancy outcome. CONCLUSION(S) Although seminal plasma GPX activity was related to semen quality according to WHO parameters, such an association was not found with IVF-intracytoplasmic sperm injection (ICSI) outcome, presumably because of the well-known ability of IVF-ICSI procedures to overcome sperm deficiencies in the fertilization process.

Doxorubicin induces ceramide and diacylglycerol accumulation in rat hepatocytes through independent routes.

Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited due to its toxicity. This toxicity has been associated with free radicals generated during the drug metabolism. We previously found that DOX increased the intracellular diacylglycerol (DAG) levels at 1h in isolated rat hepatocytes, probably by mobilizing choline-enriched phospholipids. In this work, we studied the effects of DOX on oxidative stress markers, and the possible contribution of ceramide metabolism to DAG accumulation. Other possible routes of DAG production, such as impairment of triacylglycerol (TAG) synthesis, and their connection with oxidative stress were also investigated. Time-course experiments revealed that DOX decreased intracellular GSH at 2h, but did not affect cell viability, ATP or malondialdehyde (MDA) levels at any time. DOX did not modify the intracellular levels of [(3)H]-ceramide during the first 90 min of exposure, but increased it significantly at 2h. [(3)H]-Sphingomyelin remained unchanged during the whole period. These results indicate that ceramide metabolism is not involved in the early DAG response to DOX. The drug markedly increased the incorporation of [(3)H]-oleate into intracellular DAG from 60 min. In contrast, DOX reduced the incorporation of [(3)H]-oleate into intracellular phospholipids and TAG. DOX inhibited TAG synthesis at the DAG acyltransferase step. These results suggest that DOX increases the intracellular levels of the lipid messengers, ceramide and DAG, by independent mechanisms. Activation of the de novo synthesis of ceramide is probably involved in the sphingolipid accumulation, while inhibition of TAG synthesis contributes to DAG accumulation, this response being independent of oxidative damage.