Marcelo G. Bonini

Nitrogen dioxide and carbonate radical anion: Two emerging radicals in biology

Nitrogen dioxide and carbonate radical anion have received sporadic attention thus far from biological investigators. However, accumulating data on the biochemical reactions of nitric oxide and its derived oxidants suggest that these radicals may play a role in various pathophysiological processes. These potential roles are also indicated by recent studies on the high efficiency of urate and nitroxides in protecting cells and whole animals against the injury associated with conditions of excessive nitric oxide production. The high protective effects of these antioxidants are incompletely defined at the mechanistic level but some of them can be explained by their efficiency in scavenging peroxynitrite-derived radicals, particularly nitrogen dioxide and carbonate radical anion. In this review, we provide a framework for this hypothesis and discuss the potential sources and properties of these radicals that are likely to become increasingly recognized as important mediators of biological processes.

Direct EPR Detection of the Carbonate Radical Anion Produced from Peroxynitrite and Carbon Dioxide

The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra- and extracellular compartments. Peroxynitrite anion (ONOO−) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO2 −), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO·̄3). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6–9. The radical was unambiguously identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with bicarbonate labeled with 13C. In this case, the singlet EPR signal obtained with 12C bicarbonate splits into the expected doublet because of 13C (a(13C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO·̄3). Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.

Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria

Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes.

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.

Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Caveolin-1–mediated inhibition of endothelial nitric oxide synthase signaling is essential for adherens junction integrity and endothelial permeability homeostasis.

A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunction

Telomerase is a reverse transcriptase specialized in telomere synthesis. The enzyme is primarily nuclear where it elongates telomeres, but many reports show that the catalytic component of telomerase (in humans called hTERT) also localizes outside of the nucleus, including in mitochondria. Shuttling of hTERT between nucleus and cytoplasm and vice versa has been reported, and different proteins shown to regulate such translocation. Exactly why telomerase moves between subcellular compartments is still unclear. In this study we report that mutations that disrupt the nuclear export signal (NES) of hTERT render it nuclear but unable to immortalize cells despite retention of catalytic activity in vitro. Overexpression of the mutant protein in primary fibroblasts is associated with telomere‐based cellular senescence, multinucleated cells and the activation of the DNA damage response genes ATM, Chk2 and p53. Mitochondria function is also impaired in the cells. We find that cells expressing the mutant hTERT produce high levels of mitochondrial reactive oxygen species and have damage in telomeric and extratelomeric DNA. Dysfunctional mitochondria are also observed in an ALT (alternative lengthening of telomeres) cell line that is insensitive to growth arrest induced by the mutant hTERT showing that mitochondrial impairment is not a consequence of the growth arrest. Our data indicate that mutations involving the NES of hTERT are associated with defects in telomere maintenance, mitochondrial function and cellular growth, and suggest targeting this region of hTERT as a potential new strategy for cancer treatment.

MnSOD upregulation sustains the Warburg effect via mitochondrial ROS and AMPK-dependent signalling in cancer

Manganese superoxide dismutase (MnSOD/SOD2) is a mitochondria-resident enzyme that governs the types of reactive oxygen species egressing from the organelle to affect cellular signaling. Here, we demonstrate that MnSOD upregulation in cancer cells establishes a steady flow of H2O2 originating from mitochondria that sustains AMP-activated kinase (AMPK) activation and the metabolic shift to glycolysis. Restricting MnSOD expression or inhibiting AMPK suppress the metabolic switch and dampens the viability of transformed cells indicating that the MnSOD/AMPK axis is critical in support cancer cell bioenergetics. Recapitulating in vitro findings, clinical and epidemiologic analyses of MnSOD expression and AMPK activation indicated that the MnSOD/AMPK pathway is most active in advanced stage and aggressive breast cancer subtypes. Taken together, our results indicate that MnSOD serves as a biomarker of cancer progression and acts as critical regulator of tumor cell metabolism.

Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition.

The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

Male infertility: a public health issue caused by sexually transmitted pathogens

Sexually transmitted diseases (STDs) are caused by several pathogens, including bacteria, viruses and protozoa, and can induce male infertility through multiple pathophysiological mechanisms. Additionally, horizontal transmission of STD pathogens to sexual partners or vertical transmission to fetuses and neonates is possible. Chlamydia trachomatis, Ureaplasma spp., human papillomavirus, hepatitis B and hepatitis C viruses, HIV-1 and human cytomegalovirus have all been detected in semen from symptomatic and asymptomatic men with testicular, accessory gland and urethral infections. These pathogens are associated with poor sperm quality and decreased sperm concentration and motility. However, the effects of these STD agents on semen quality are unclear, as are the effects of herpes simplex virus type 1 and type 2, Neisseria gonorrhoeae, Mycoplasma spp., Treponema pallidum and Trichomonas vaginalis, because few studies have evaluated the influence of these pathogens on male infertility. Chronic or inadequately treated infections seem to be more relevant to infertility than acute infections are, although in many cases the exact aetiological agents remain unknown.

Inhibition of c-Src tyrosine kinase prevents angiotensin II-mediated connexin-43 remodeling and sudden cardiac death.

OBJECTIVES The aim of this study was to test whether c-Src tyrosine kinase mediates connexin-43 (Cx43) reduction and sudden cardiac death in a transgenic mouse model of cardiac-restricted overexpression of angiotensin-converting enzyme (ACE8/8 mice). BACKGROUND Renin-angiotensin system activation is associated with an increased risk for arrhythmia and sudden cardiac death, but the mechanism is not well understood. The up-regulation of c-Src by angiotensin II may result in the reduction of Cx43, which impairs gap junction function and provides a substrate for arrhythmia. METHODS Wild-type and ACE8/8 mice with and without treatment with the c-Src inhibitor 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP1) were studied. Telemetry monitoring, in vivo electrophysiologic studies, Western blot analyses for total and phosphorylated c-Src and Cx43, immunohistochemistry staining for Cx43, and functional assessment of Cx43 with fluorescent dye diffusion were performed. RESULTS The majority of the arrhythmic deaths resulted from ventricular tachycardia degenerating to ventricular fibrillation (83%). Levels of total and phosphorylated c-Src were increased and Cx43 reduced in ACE8/8 mice. PP1 reduced total and phosphorylated c-Src levels, increased Cx43 level by 2.1-fold (p < 0.005), increased Cx43 at the gap junctions (immunostaining), improved gap junctional communication (dye spread), and reduced ventricular tachycardia inducibility and sudden cardiac death. The survival rate increased from 11% to 86% with 4 weeks of PP1 treatment (p < 0.005). Treatment with an inactive analog did not change survival or Cx43 levels. CONCLUSIONS Renin-angiotensin system activation is associated with c-Src up-regulation, Cx43 loss, reduced myocyte coupling, and arrhythmic sudden death, which can be prevented by c-Src inhibition. This suggests that an increase in c-Src activity may help mediate renin-angiotensin system-induced arrhythmias and that c-Src inhibitors might exert antiarrhythmic activity.