Differential nitros(yl)ation of blood and tissue constituents during glyceryl trinitrate biotransformation in vivo

Nitric oxide (NO)-derived products may modify tissue constituents, forming S- and N-nitroso adducts and metal nitrosyls implicated in NO signaling. Nitrovasodilator drugs have been in widespread use for more than a century, yet their biotransformation pathways to NO and their effects as NO donors across tissues remain ill defined. By using a metabonomics approach (termed "NObonomics") for detailing the global NO-related metabolism of the cornerstone nitrovasodilator, glyceryl trinitrate (GTN; 0.1-100 mg/kg), in the rat in vivo, we find that GTN biotransformation elicits extensive tissue nitros(yl)ation throughout all major organ systems. The corresponding reaction products remained detectable hours after administration, and vascular tissue was not a major nitros(yl)ation site. Extensive heart and liver modifications involved both S- and N-nitrosation, and RBC S-nitrosothiol formation emerged as a sensitive indicator of organic nitrate metabolism. The dynamics of GTN-derived oxidative NO metabolites in blood did not reflect the nitros(yl)ation patterns in the circulation or in tissues, casting doubt on the usefulness of plasma nitrite/nitrate as an index of NO/NO-donor biodynamics. Target-tissue NO metabolites varied in amount and type with GTN dose, suggesting a dose-sensitive shift in the prevailing routes of GTN biotransformation ("metabolic shunting") from thiol nitrosation to heme nitrosylation. We further demonstrate that GTN-induced nitros(yl)ation is modulated by a complex, tissue-selective interplay of enzyme-catalyzed pathways. These findings provide insight into the global in vivo metabolism of GTN at pharmacologically relevant doses and offer an additional experimental paradigm for the NObonomic analysis of NO-donor metabolism and signaling.     

Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.     

Multipronged evaluation of genotoxicity in Indian petrol-pump workers

Petrol (gasoline) contains a number of toxicants. This study used human biomonitoring to evaluate the genotoxic effects of exposure to benzene in petrol fumes in 100 Indian petrol-pump workers (PPWs) and an equal number of controls. The study was corroborated with in silico assessments of the Comet assay results from the human biomonitoring study. An in vitro study in human lymphocytes was also conducted to understand the genotoxicity of benzene and its metabolites. In a subset of the population studied, higher blood benzene levels were detected in the PPWs (n = 39; P < 0.01) than the controls (n = 18), and 100-250 ppb benzene was also detected in air samples from the petrol pumps. PPWs had higher levels of DNA damage than the controls (P < 0.01). In addition, the micronucleus assay was performed on lymphocytes from a subset of the subjects, and the micronucleus frequency for PPWs was significantly higher (n = 39; 14.79 +/- 3.92 per thousand) than the controls (n = 18; 7.54 +/- 3.00 per thousand). Human lymphocytes were treated in vitro with benzene and several of its metabolites and assayed for DNA damage with the Comet assay. Benzene and its metabolites produced significant (P < 0.05) levels of DNA damage at and above concentrations of 10 microM. The metabolite, p-benzoquinone, produced the greatest amount of DNA damage, followed by hydroquinone > benzene > catechol > 1,2,4,-benzenetriol > muconic acid. This study demonstrates that, using sensitive techniques, it is possible to detect human health risks at an early stage when intervention is possible. possible.     

Dorsal lunate tilt (DISI configuration): sign of scaphoid fracture displacement

Excessive dorsiflexion (dorsal tilting) of the lunate on a lateral wrist radiograph can be an important sign of carpal injury. Lunate dorsiflexion is a well-recognized sign of an intercarpal ligamentous injury pattern known as dorsal intercalated segment instability (DISI). It is less well recognized that excessive dorsal tilting of the lunate (DISI configuration) can also be produced by displacement of a scaphoid waist fracture. Since the management and prognosis of displaced scaphoid fractures may be quite different from those for nondisplaced fractures, radiologists can make an important contribution by recognizing dorsal tilting of the lunate and appreciating that it may be an important, indirect sign of scaphoid fracture displacement, which may not be directly visualized with standard wrist radiography. In this setting, computed tomography or complex motion tomography may be helpful for further evaluation of the scaphoid fracture.     

Neurotization improves contractile forces of tissue-engineered skeletal muscle

Engineered functional skeletal muscle would be beneficial in reconstructive surgery. Our previous work successfully generated 3-dimensional vascularized skeletal muscle in vivo. Because neural signals direct muscle maturation, we hypothesized that neurotization of these constructs would increase their contractile force. Additionally, should neuromuscular junctions (NMJs) develop, indirect stimulation (via the nerve) would be possible, allowing for directed control. Rat myoblasts were cultured, suspended in fibrin gel, and implanted within silicone chambers around the femoral vessels and transected femoral nerve of syngeneic rats for 4 weeks. Neurotized constructs generated contractile forces 5 times as high as the non-neurotized controls. Indirect stimulation via the nerve elicited contractions of neurotized constructs. Curare administration ceased contraction in these constructs, providing physiologic evidence of NMJ formation. Histology demonstrated intact muscle fibers, and immunostaining positively identified NMJs. These results indicate that neurotization of engineered skeletal muscle significantly increases force generation and causes NMJs to develop, allowing indirect muscle stimulation.     

DNA damage induced by industrial solid waste leachates in Drosophila melanogaster: a mechanistic approach

Genomic stability requires that error-free genetic information be transmitted from generation to generation, a process that is dependent upon efficient DNA repair. Industrial leachates which contain mixtures of diverse chemicals are a major environmental concern. The interaction between these chemicals may have synergistic, antagonistic, or simply additive effects on biological systems. In the present study, the Comet assay was used to measure the DNA damage produced by leachates of solid wastes from flashlight battery, pigment, and tanning factories in the midgut cells and brain ganglia of Drosophila melanogaster mutants deficient in DNA repair proteins. Larvae were allowed to feed for 48 or 72 hr on diets containing 0.1, 0.5, and 2.0% (v/v) of the leachates. Physicochemical analysis run on the solid wastes, leachates, and treated larvae detected elevated levels of heavy metals. Leachates produced significantly greater levels of DNA damage in mutant strains mei41 (deficient in cell cycle check point protein), mus201 (deficient in excision repair protein), mus308 (deficient in postreplication repair protein), and rad54 (deficient in double strand break repair protein) than in the OregonR(+) wild-type strain. Larvae of the ligaseIV mutant (deficient in double strand break repair protein) were hypersensitive only to the pigment plant waste leachate. Conversely, the dnase2 mutant (deficient in protein responsible for degrading fragmented DNA) was more sensitive to DNA damage induction from the flashlight battery and tannery waste leachates. Our data demonstrate that repair of DNA damage in organisms exposed to leachates is dependent upon several DNA repair proteins, indicative of the involvement of multiple overlapping repair pathways. The study further suggests the usefulness of the Comet assay for studying the mechanisms of DNA repair in Drosophila.