Peroxynitrite-Dependent Aromatic Hydroxylation and Nitration of Salicylate and Phenylalanine. Is Hydroxyl Radical Involved?

There is considerable dispute about whether the hydroxylating ability of peroxynitrite (ONOO^-)-derived species involves hydroxyl radicals (OH*). This was investigated by using salicylate and phenylalanine, attack of OH* upon which leads to the formation of 2, 3- and 2, 5-dihydroxybenzoates, and o-, m- and p-tyrosines respectively. On addition of ONOO- to salicylate, characteristic products of hydroxylation (and nitration) were observed in decreasing amounts with rise in pH, although added products of hydroxylation of salicylate were not recovered quantitatively at pH 8.5, suggesting further oxidation of these products and underestimation of hydroxylation at alkaline pH. Hydroxylation products decreased in the presence of several OH* scavengers, especially formate, to extents similar to those obtained when hydroxylation was achieved by a mixture of iron salts, H₂O₂ and ascorbate. However, OH* scavengers also inhibited formation of salicylate nitration products. Ortho, p- and m-tyrosines as well as nitration products were also observed when ONOO^- was added to phenylalanine. The amounts of these products again decreased at high pH and were decreased by addition of OH* scavengers. We conclude that although comparison with Fenton systems suggests OH* formation, simple homolytic fission of peroxynitrous acid (ONOOH) to OH* and NO₂ would not explain why OH* scavengers inhibit formation of nitration products.     

Antioxidant Properties of S-Adenosyl-l-Methionine: A Proposed Addition to Organ Storage Fluids

Glutathione (GSH) depletion adversely affects the survival of organ grafts. Supplementation of commercial organ preservation solutions with GSH is complicated by the ease of oxidation of its thiol group and its ability to act as a pro-oxidant under certain conditions. Alternative sulphur-containing compounds such as S-adenosyl-

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-methionine (SAM) can reduce ischaemia-reperfusion injury, possibly by acting as glutathione precursors, and are effective when added to preservation solutions. Although the antioxidant properties of GSH are known in some detail, there is little information on the ability of SAM to interact directly with reactive oxygen species (ROS) produced during ischaemia-reperfusion injury. This work compares the interaction of SAM and GSH with several ROS which may be formed during ischaemia-reperfusion. In a variety of lipid peroxidation systems, SAM and GSH had little effect except at high concentrations (5 mM) where they became pro-oxidant. Scavenging of O₂^˙− by both species was slow. SAM was less effective than GSH at preventing damage by peroxynitrite or HOCl. In contrast, SAM was more effective than GSH in scavenging hydroxyl radicals (^˙OH) and in chelating iron ions to inhibit ^˙OH generation. Unlike GSH, SAM did not stimulate ^˙OH formation at low concentrations. The beneficial effects of SAM in preservation solutions could therefore include direct radical scavenging as well as acting as a precursor for intracellular GSH.     

A reevaluation of the peroxynitrite scavenging activity of some dietary phenolics

Peroxynitrite is implicated in many diseases. Hence, there is considerable interest in potential therapeutic peroxynitrite scavengers. Diet-derived phenolics have been claimed to be powerful peroxynitrite scavengers. However, the reactivity of peroxynitrite can be significantly modified by bicarbonate and this has not been considered in evaluations of the scavenging activity of phenols. Bicarbonate (25 mM) significantly decreased the ability of several phenolic compounds (caffeic acid, o- and p-coumaric acid, gallic acid, ferulic acid) but not others (catechin and epicatechin) to inhibit peroxynitrite-mediated tyrosine nitration. Bicarbonate (25 mM) also decreased the ability of catechin, epicatechin, quercetin and ferulic acid but not chlorogenic acid, gallic acid, caffeic acid and o-coumaric acid to inhibit peroxynitrite-mediated alpha(1)-antiproteinase inactivation. These results show that physiological concentrations of bicarbonate substantially modify the ability of dietary phenolics to prevent peroxynitrite-mediated reactions. When assessing compounds for peroxynitrite scavenging, experiments should be conducted in the presence of bicarbonate to avoid misleading results.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

Prepregnancy Obesity and Risk of Stillbirth in Viable Twin Gestations

We sought to estimate the impact of prepregnancy obesity on demise of one or both fetuses in twin gestations. We performed a retrospective cohort study using the Missouri maternally linked cohort files (years 1989–2005). Prepregnancy obesity was defined as a BMI ≥30. Outcomes of interest were stillbirth (intrauterine fetal death at ≥20 weeks' gestation) and demise of one (partial loss) or both (complete loss) fetuses, regardless of the cause. We used Cox Proportional Hazards with correction for intracluster correlation to obtain risk estimates. The overall stillbirth rate for twin gestations was 15.5/1,000 (18.4/1,000 vs. 14.5/1,000 in obese and normal weight mothers, respectively; P = 0.02). The rate for complete fetal loss was higher in obese mothers (8.3/1,000 vs. 5.6/1,000; P = 0.01) but was comparable for partial fetal loss (19.1/1,000 for obese vs. 16.3/1,000 for normal weight mothers; P = 0.1). Adjusted estimates confirmed these findings (adjusted hazards ratio (AHR) and 95% confidence interval (CI) = 1.31 (1.02–1.68) for stillbirth; AHR = 1.59; CI = 1.01–2.51) for complete loss; and AHR = 1.21; CI = 0.91–1.62) for partial loss. Subanalysis conducted on stillbirth showed that the risk associated with obesity was only elevated for same‐sex (AHR = 1.54; CI = 1.15–2.04) but not opposite‐sex twins (0.99; CI = 0.56–1.75). Our findings may find utility in counseling of obese women with twin gestations.     

Social monogamy in the cleaning goby Elacatinus evelynae: ecological constraints or net benefit?

We tested predictions from four hypotheses to explain the occurrence of long-term socially monogamous pairs in the Caribbean cleaning goby Elacatinus (=Gobiosoma) evelynae, namely (1) resource limitation, (2) low population and/or low mate density, (3) territorial defence and (4) net benefit of single-mate sequestration. We found no evidence that resources, in terms of available cleaning stations or clients to clean, were limited (1) or that after experimental goby removals, single individuals could not maintain cleaning stations alone (2). Population density was low but this did not prevent artificially widowed fish from remating quickly with individuals as large as their initial partners (3). Social monogamy in E. evelynae appears to result from the benefits associated with sequestering a large, high-quality mate (4). Both males and females showed intrasexual aggression towards experimental intruders consistent with mate guarding. Opportunities for polygynous matings by males, assessed by comparing the sizes, distances between and mating synchrony of neighbouring pairs, appeared both low and of limited value. Males therefore benefit most from guarding a larger, more fecund female. Females spent longer cleaning when paired with a large male, indicating that the benefits of guarding a high-quality mate may extend outside of the reproductive period for socially monogamous species. These results add to an increasing number of studies on coral reef fish showing mate-guarding behaviour and benefits to males and females from sequestering a single mate. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.