Hydroxymethylglutaryl-CoA reductase inhibitors (statins) for the treatment of sepsis in adults - A systematic review and meta-analysis.

OBJECTIVES: The pleiotropic effect of hydroxymethylglutaryl-CoA reductase inhibitors (statins) might have a beneficial effect in sepsis through several mechanisms. The aim was to assess the efficacy and safety of statins, compared with placebo, for the treatment of sepsis in adults. METHODS: We searched the following databases: the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, 2017, Issue 12), OVID MEDLINE (from 1966 to January 2018), Embase (Ovid SP, from 1974 to January 2018), and LILACS (from 1986 to January 2018). We also searched the trial registries ISRCTN and ClinicalTrials.gov to January 2018. The eligibility criteria were randomized controlled trials comparing the treatment of statins versus placebo in adult patients who were hospitalized due to sepsis. Participants were adults (16 years and older) hospitalized because of sepsis or who developed sepsis during admission. Interventions were treatment with hydroxymethylglutaryl-CoA reductase inhibitors (statins) versus no treatment or placebo. We performed a systematic review of all randomized controlled trials published until January 2018, assessing the efficacy and safety of statins in sepsis treatment. Two primary outcomes were assessed: 30-day overall mortality and deterioration to severe sepsis during management. Secondary outcomes were hospital mortality, need for mechanical ventilation and drug related adverse events. RESULTS: Fourteen trials evaluating 2628 patients were included. Statins did not reduce 30-day all-cause mortality neither in all patients (risk ratio (RR) 0.96, 95% confidence interval (CI) 0.83-1.10), nor in a subgroup of patients with severe sepsis (RR 0.97, 95% CI 0.84-1.12). The certainty of evidence for both outcomes was high. There was no change in the rate of adverse events between study arms (RR 1.24, 95% CI 0.94 to 1.63). The certainty of evidence for this outcome was high. CONCLUSIONS: The use of statin therapy in adults for the indication of sepsis is not recommended.

Role of the androgen receptor CAG repeat polymorphism in prostate cancer, and spinal and bulbar muscular atrophy.

Androgens are involved in the development of several tissues, including prostate, skeletal muscle, bone marrow, hair follicles, and brain. Most of the biological effects of the androgens are mediated through an intracellular transcription factor, the androgen receptor (AR) at the level of gene regulation. Several types of mutations in the AR gene have been linked to endocrine dysfunctions. The expansion of CAG codon repeat, coding for a polyglutamine (PolyQ) tract in the N-terminal domain is one such mutation. The polyQ chain length impacts AR's ability to interact with critical coregulators, which in turn modulates its transcriptional efficacy. Pathologic manifestations of variations in polyQ chain length have been associated with prostate cancer susceptibility, and the Spinal and Bulbar Muscular Atrophy (SBMA), a neurodegenerative disease. In this review article, we discuss multiple aspects of the role of polyQ chain length in the actions of the AR, their importance in prostate cancer development and progression, and SBMA with an aim to understand the underlying mechanisms involved in these diseases, which can be targeted for future therapeutic approaches.

Heme deficiency selectively interrupts assembly of mitochondrial complex IV in human fibroblasts: revelance to aging.

Heme deficiency was studied in young and old normal human fibroblasts (IMR90). Regardless of age, heme deficiency increased the steady-state level of oxidants and lipid peroxidation and sensitized the cells to fluctuations in intracellular Ca(2+). Heme deficiency selectively decreased the activity and protein content of mitochondrial complex IV (cytochrome c oxidase) by 95%, indicating a decrease in successful assembly. Complexes I-III and catalase remained intact under conditions of heme deficiency, whereas ferrochelatase was up-regulated. Complex IV is the only hemeprotein in the cell that contains heme a, which may account for its susceptibility. The rate of removal and assembly of complex IV declines with age. These findings are relevant to worldwide iron deficiency in women and children and to an age-related decline in complex IV in Alzheimer's disease patients.

N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro.

N-t-butyl hydroxylamine (NtBHA) delays senescence-dependent changes in human lung fibroblasts (IMR90) (Atamna et al., J. Biol. Chem. 275, 6741-6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary fibroblasts (IMR90). In NtBHA-treated rats, the age-dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence-associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of growth factors. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10- to 15-fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical-induced toxicity to mitochondria.

N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts.

Alpha-phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice. N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PBN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 microM compared with 200 microM PBN to produce a similar effect, suggesting that N-t-butyl hydroxylamine is the active form of PBN. N-Benzyl hydroxylamine and N-methyl hydroxylamine compounds unrelated to PBN were also effective in delaying senescence, suggesting the active functional group is the N-hydroxylamine. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants, as measured by the oxidation of 2', 7'-dichlorodihydrofluorescin and the increase in the GSH/GSSG ratio. The acceleration of senescence induced by hydrogen peroxide is reversed by the N-hydroxylamines. DNA damage, as determined by the level of apurinic/apyrimidinic sites, also decreased significantly following treatment with N-hydroxylamines. The N-hydroxylamines appear to be effective through mitochondria; they delay age-dependent changes in mitochondria as measured by accumulation of rhodamine-123, they prevent reduction of cytochrome C(FeIII) by superoxide radical, and they reverse an age-dependent decay of mitochondrial aconitase, suggesting they react with the superoxide radical.

A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

The malaria parasite supplies glutathione to its host cell--investigation of glutathione transport and metabolism in human erythrocytes infected with Plasmodium falciparum.

Malaria-infected red blood cells are under a substantial oxidative stress. Glutathione metabolism may play an important role in antioxidant defense in these cells, as it does in other eukaryotes. In this work, we have determined the levels of reduced and oxidized glutathione (GSH and GSSG, respectively) and their distributions in the parasite, and in the host-cell compartments of human erythrocytes infected with the malaria parasite Plasmodium falciparum. In intact trophozoite-infected erythrocytes, [GSH] is low and [GSSG] is high, compared with the levels in normal erythrocytes. Normal erythrocytes and the parasite compartment display high GSH/GSSG ratios of 321.6 and 284.5, respectively, indicating adequate antioxidant defense. This ratio drops to 26.7 in the host-cell compartment, indicating a forceful oxidant challenge, the low ratios resulting from an increase in GSSG and a decline in GSH concentrations. On the other hand, the concentrations of GSH and GSSG in the parasite compartment remain physiological and comparable to their concentrations in normal red blood cells. This results from de novo glutathione synthesis and its recycling, assisted by the intensive activity of the hexose monophosphate shunt in the parasite. A large efflux of GSSG from infected cells has been observed, its rate being similar from free parasites and from intact infected cells. This result suggests that de novo synthesis by the parasite is the dominating process in infected cells. GSSG efflux from the intact infected cell is more than 60-fold higher than the rate observed in normal erythrocytes, and is mediated by permeability pathways that the parasite induces in the erythrocyte's membrane. The main route for GSSG efflux through the cytoplasmic membrane of the parasite seems to be due to a specific transport system and occurs against a concentration gradient. Gamma-glutamylcysteine [Glu(-Cys)] and GSH can penetrate through the pathways from the extracellular space into the host cytosol, but not into that of the parasite. This implies that the parasite membrane is impermeable to these peptides, and that the host cannot supply GSH to the parasite as suggested previously. Exogenous Glu(-Cys) is not converted into GSH in the host cell, arguing that GSH synthetase may not be functional. Compartment analysis of Mg2+ in infected erythrocytes revealed that the host compartment exhibits a low concentration of Mg2+ (0.5 mM) in comparison with the parasite compartment (4 mM) and the normal erythrocytes (1.5-3 mM). The drop in [Mg2+] results in cessation of Glu(-Cys) synthesis, and hence of GSH synthesis in the host-cell compartment. The decrease in [Mg2+] can affect other Mg2+-ATP-dependent functions, such as Na+ and Ca2+ active efflux. The present investigation confirms that the host-cell compartment is oxidatively distressed, whereas the parasite is efficiently equipped with anti-oxidant means that protect the parasite from the oxidative injury. The parasite has a huge capacity for de novo synthesis of GSH and for the reduction of GSSG. Part of the GSSG that is actively extruded from the parasite is reduced to GSH in the host cell whose own GSH synthesis is crippled.

Resistance of glucose-6-phosphate dehydrogenase deficiency to malaria: effects of fava bean hydroxypyrimidine glucosides on Plasmodium falciparum growth in culture and on the phagocytosis of infected cells.

The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malarial combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their beta-O-glucosidic bond, became protein pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of beta-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype.

Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo.

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.

Heme degradation in the presence of glutathione. A proposed mechanism to account for the high levels of non-heme iron found in the membranes of hemoglobinopathic red blood cells.

Unstable hemoglobins and oxidative conditions tend to produce hemichromes which demonstrably release their heme to the erythrocyte membrane, with consequent lipid peroxidation and cell lysis. High levels of non-heme iron are also found in such circumstances, but the origin of this iron is uncertain. In the present work, we show that reduced glutathione (GSH) is able to degrade heme in solution with a pH optimum of 7. Degradation depended on the presence of oxygen and on heme and GSH concentrations. It was inhibited by catalase and superoxide dismutase, implicating the involvement of perferryl reactive species in the process of heme degradation. Heme degradation at pH 7 and 37 degrees C is rapid (t1/2 = 70 s) and results in the release of iron from heme. Heme that was dissolved in red blood cell ghosts is also degraded by GSH with a concomitant increase in non-heme iron, most of which (75%) remains associated with the cell membrane. Loading of intact erythrocytes with heme was followed by time-dependent decrease of membrane-associated heme and caused an acceleration of the hexose monophosphate shunt due to the production of H2O2 and the oxidation of intracellular GSH. Most of the activation of the hexose monophosphate pathway was due to redox cycling of iron, since iron chelators inhibited it considerably. These results explain the origin of non-heme iron found in the membrane of sickle cells and the oxidative stress that is observed in these and other abnormal erythrocytes.

Hexose-monophosphate shunt activity in intact Plasmodium falciparum-infected erythrocytes and in free parasites.

The hexose monophosphate shunt (HMS) produces NADPH for reductive antioxidant protection and for metabolic regulation, as well as ribose-5-phosphate needed for the synthesis of nucleic acids. Since malaria-infected red blood cells (RBC) are under endogenous oxidant stress, it was interesting to determine HMS activity in intact infected cells, as well as in free parasites. HMS activity was determined by measuring the evolution of 14CO2 from D-[1-14C]glucose in normal RBC, in intact Plasmodium falciparum-infected RBC (IRBC) and in free parasites. The HMS activity of IRBC was found to be 78 times higher than that of normal RBC. This activity increased with parasite maturation from the ring stage toward the trophozoite stage, and declined at the schizont stage. The HMS activity of the parasite contributes 82% of the total observed in the intact IRBC, and that of the host cell is increased some 24-fold. The increased reducing capacity of IRBC and free parasites were also evidenced by the larger ability for reductive accumulation of methylene blue. Since the endogenous oxidative stress is produced by the parasite digestion of the host cell's hemoglobin, inhibition of this process with protease inhibitors, by alkalinization of the parasite's food vacuole, or by the application of antimalarial drugs, resulted in 20-44% inhibition of IRBC HMS activity. A similar inhibition was observed in the presence of scavengers of oxidative radicals, uric and benzoic acids. These inhibitors had only a minor effect on the HMS activity of free parasites. D-[1-14C]glucose and D-[6-14C]glucose contributed equally to newly synthesized nucleic acids, suggesting that ribose-5-phosphate needed for this synthesis is contributed by the non-oxidative activity of HMS. These results imply that a major portion of parasite HMS activity and the activated HMS of the host cell are devoted to counteract the endogenously generated oxidative stress.

The redox status of malaria-infected erythrocytes: an overview with an emphasis on unresolved problems.

The various mechanisms involved in the redox defence of normal erythrocytes are adequately known. They are herein briefly reviewed, outlining the principal enzymes and metabolic pathways, such as superoxide dismutase, catalase, glutathione peroxidase and reductase, the hexose monophosphate shunt (HMS) and glutathione synthesis and turnover. The intraerythrocytic malaria parasite is imposing an oxidative stress on its host cell. Malaria infected cells produce O2-, H2O2, enhance lipide peroxidation and activate host cell HMS. This stress is produced during the digestion of host cell hemoglobin by the parasite. Hence, both parasite and host cell must be able to confront this stress. The antioxidant defence systems of the parasite and the response of those systems in the infected host cell are reviewed, underscoring unresolved problems. Nothing is virtually known on the parasite's glutathione metabolism, and on possible interactions between host cell and parasite antioxidant defence systems. The postulate that 1. host cell activated HMS in conjunction with purine salvage can provide purine nucleotides to the parasite, and 2. that glutathione transferase can participate in parasite resistance to antimalarial drugs, are also discussed.

Origin of reactive oxygen species in erythrocytes infected with Plasmodium falciparum.

Oxidative radicals are demonstrably produced in malaria-infected erythrocytes. In order to verify the biochemical origin of these radicals, erythrocyte lysate was brought to acid pH to mimic the environment of the parasite food vacuole into which host cell cytosol is transferred during parasite feeding. Oxyhemoglobin, but not deoxyhemoglobin, is rapidly converted to methemoglobin at rates which decline with increasing pH. The rate of conversion is further increased in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and the extent of inhibition of the lysate catalase increases upon acidification, implying that H2O2 is thus produced by the spontaneous dismutation of superoxide radicals generated during methemoglobin formation. Intact Plasmodium falciparum trophozoite-infected human red blood cells (TRBC) were shown to produce H2O2 and OH radicals about twice as much as normal erythrocytes, as evidenced by the inhibition of endogenous catalase activity in the presence of 3-AT and the degradation of deoxyribose, respectively. Increased H2O2 levels and catalase activity were found in both host cell and parasite compartments. No increase in H2O2 production over that observed in uninfected erythrocytes could be detected at the ring stage when host cell digestion is absent. H2O2 and OH radicals production in TRBC was considerably reduced when digestion of host cell cytosol was inhibited either by antiproteases (which reduce the proteolysis of imported catalase) or by its alkalinization with NH4Cl (which reduce methemoglobin formation). These results suggest that reactive oxygen species are produced in the parasite's food vacuole during the digestion of host cell cytosol, and are able to egress from the parasite to the host cell compartment.