Ethanol-Induced Enhancement of Cocaine Bioactivation and Irreversible Protein Binding: Evidence Against a Role of Cytochrome P-450IIE1

Chronic ethanol consumption potentiates cocaine-induced liver injury in rodents. Since cocaine has to be bioactivated by a cytochrome P-450-dependent N-oxidative pathway to exert its hepatotoxic effects, we studied the role of the ethanol-inducible P-450IIE1 for cocaine metabolism. Male Sprague-Dawley rats were pretreated with either a liquid diet containing ethanol (30% of calories) for 4 weeks or injected with pyrazole (200 mg/kg/day, ip, for 3 days). Both agents induced microsomal p-nitrophenol hydroxylation which is a probe for the catalytic activity of P-450IIE1. However, only ethanol, but not pyrazole, increased both microsomal cocaine N-demethylase activity (by 47%) and the extent of irreversible binding of [3H]-cocaine to microsomal proteins (by 100%), which was taken as a quantitative endpoint for the formation of a reactive metabolite. Cocaine N-demethylation and irreversible protein binding of cocaine were not inhibited by P-450IIE1 isozyme-selective substrates, nor was the rate of cocaine metabolism and binding decreased by functionally active polyclonal anti-rat P-450IIE1 antibodies. Furthermore, pyrazole pretreatment sensitized cultured hepatocytes to the glutathione-dependent cytotoxic effects of nontoxic concentrations of cocaine. These results indicate that (a) cocaine is not a major substrate for the ethanol-inducible P-450IIE1, (b) the enhancing effects of ethanol on cocaine bioactivation may be due to induction of other P-450 isoforms, and (c) induction of P-450IIE1 may potentiate cocaine-induced hepatocellular toxicity in vitro independently of cocaine metabolism, e.g., by P-450IIE1-dependent oxidative stress.     

Protection of the ischemic myocardium from reperfusion injury by prostaglandin E1 inhibition of ischemia-induced neutrophil activation

Summary This study investigates the action of intravenous PGE₁ on myocardial reperfusion injury and the possible involvement of antineutrophil activities. Cats were subjected to 3 h of temporary ligation of the left anterior descending coronary artery, followed by 2 h of reperfusion. Animals were treated with PGE₁ (5 g/kg x min) or vehicle (saline solution), starting 0.5 h after coronary artery occlusion. Vehicle-treated cats exhibited a significant loss of cardiac creatine phosphokinase specific activity at 5 h, accompanied by a significant ischemia-induced rise in the ST segment of the ECG and development of a Q wave after starting reperfusion. All of these alterations were largely prevented by PGE₁ treatment. PGE₁ exerted some blood-pressurelowering activity at 5 h (P > 0.05) but did not reduce myocardial contractile force and oxygen consumption. PGE₁ modestly antagonized ischemia-induced formation of platelet aggregates. However, PGE₁ prevented the rise in peripheral white blood cell count during ischemia and reperfusion and inhibited the generation of reactive oxygen species (myeloperoxidase assay) from zymosan-stimulated whole blood ex vivo. The ratio of generation of reactive oxygen species/white blood count remained unchanged. It is concluded that PGE₁ protects the ischemic myocardium from acute reperfusion injury and that this effect involves an action of the compound on neutrophils, probably by improved myocardial tissue preservation, resulting in reduced formation of chemotactic products and, consequently, less local neutrophil accumulation and release of noxious metabolites.Parts of these results have been presented to the 29th Spring meeting of the Deutsche Gesellschaft für Pharmakologie und Toxikologie, Mainz, 1988 Send offprint requests to K. Schrör at the above address     

Amyloid in surgical pathology

Amyloid is defined as a proteinaceous tissue deposit that shows a typical green birefringence in polarized light after staining with Congo red, the presence of non-branching linear fibrils of indefinite length with a mean diameter of 10 nm, and a distinct X-ray diffraction pattern consistent with Pauling's model of a cross -fibril. Amyloid may deposit locally or may present as a systemic disease. The origin of amyloid is diverse: 25 different fibril proteins have been described so far. The precursor proteins differ from each other in their primary structures and functions. The only common denominator is the propensity to form anti-parallel cross -fibrils under certain circumstances. Early diagnosis of amyloid is still a major challenge in surgical pathology. Histological proof can be obtained using Congo-red staining and polarization microscopy. However, small deposits may be difficult to discern, and sensitivity can be improved using fluorescence microscopy. Classification of amyloid is mandatory, since amyloid is treatable and different treatment regimens are applied to different amyloid diseases. This review focuses on the epidemiology, clinical features, pathology and diagnosis of amyloid in surgical pathology.     

An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.     

Regulatory effects of biophysical strain on rat TMJ discs

Recent studies have revealed that dynamic biomechanical forces can exert antiinflammatory and antiproteolytic effects on fibrocartitage. Whether the effects of mechanical strain also involve stimulation of the insulin-like growth factor (IGF) system and, therefore, of growth and repair of fibrocartilage has yet to be determined. The objective of this in vitro study was to determine if continuous biophysical strain regulates the gene expression of IGF1, IGF2, IGF1 receptor (IGF1R), insulin receptor substrate (IRS1), and IGF-binding proteins (IGFBP) 3 and 5 in cells from the fibrocartilaginous disc of the temporomandibular joint (TMJ). Rat TMJ disc cells were subjected to continuous biophysical strain (3% and 20%) for 4 and 24 h. Subsequently, RNA was extracted and real-time PCR was performed using an iCycler iQ detection system to analyze the gene expression of the IGF system. The gene expression of IGF1, IGF2, IGF1R, IRS1, IGFBP3, and IGFBP5 was significantly (p < 0.05) inhibited when cells were subjected to continuous biophysical strain, as compared to control at both time points. High strain induced a stronger inhibition of these molecules as compared to strain of Low magnitude. In conclusion, continuous biophysical strain seems to downregulate the expression of the IGF system and may, therefore, reduce the potential of fibrocartilage for growth and repair.     

Complement-opsonized HIV: the free rider on its way to infection

The complement system (C) is one of the main humoral components of innate immunity. Three major tasks of C against invading pathogens are: (i) lysis of pathogens by the formation of the membrane attack complex (MAC); (ii) opsonization of pathogens with complement fragments to favor phagocytosis; and (iii) attraction of inflammatory cells by chemotaxis. Like other particles, HIV activates C and becomes opsonized. To escape complement-mediated lysis, HIV has adopted various properties, which include the acquisition of HIV-associated molecules (HAMs) belonging to the family of complement regulators, such as CD46, CD55, CD59, and the interaction with humoral regulatory factors like factor H (fH). Opsonized virus may bind to complement receptor positive cells to infect them more efficiently or to remain bound on the surface of such cells. In the latter case HIV can be transmitted to cells susceptible for infection. This review discusses several aspects of C-HIV interactions and provides a model for the dynamics of this process.     

Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS

Neurotrophin treatment has so far failed to prolong the survival of individuals affected with amyotrophic lateral sclerosis (ALS), an incurable motoneuron degenerative disorder. Here we show that intracerebroventricular (i.c.v.) delivery of recombinant vascular endothelial growth factor (Vegf) in a SOD1(G93A) rat model of ALS delays onset of paralysis by 17 d, improves motor performance and prolongs survival by 22 d, representing the largest effects in animal models of ALS achieved by protein delivery. By protecting cervical motoneurons, i.c.v. delivery of Vegf is particularly effective in rats with the most severe form of ALS with forelimb onset. Vegf has direct neuroprotective effects on motoneurons in vivo, because neuronal expression of a transgene expressing the Vegf receptor prolongs the survival of SOD1(G93A) mice. On i.c.v. delivery, Vegf is anterogradely transported and preserves neuromuscular junctions in SOD1(G93A) rats. Our findings in preclinical rodent models of ALS may have implications for treatment of neurodegenerative disease in general.