Joseph A. Awad

REGULATION OF EICOSANOID PRODUCTION AND MITOGENESIS IN RAT INTESTINAL EPITHELIAL CELLS BY TRANSFORMING GROWTH FACTOR-ALPHA AND PHORBOL ESTER

Growth factors and tumor promoters have been shown to play a role in intestinal epithelial growth regulation and transformation. In this study, transforming growth factor-alpha (TGF alpha) and the tumor promoter, tetradecanoyl phorbol acetate (TPA), are shown to stimulate the production of eicosanoids by rat intestinal epithelial (RIE-1) cells in culture. A 4.5-kb mRNA, which hybridizes to the mouse cyclooxygenase-2 cDNA probe, is elevated 18-fold within 30 min after TGF alpha or TPA treatment. Stimulation of RIE-1 cells with TGF alpha leads to the increase of a protein (M(r) approximately 69,000), which binds a monospecific antibody to the mouse cyclooxygenase-2 protein. Dexamethasone markedly inhibits the increase of the 4.5-kb mRNA. Pretreatment of TGF alpha or TPA-stimulated RIE-1 cells with dexamethasone or cyclooxygenase inhibitors prevents the increase in eicosanoid production by these cells. Treatment of quiescent RIE-1 cells with TGF alpha stimulates mitogenesis. This mitogenic activity is blocked by pretreating the cells with dexamethasone or cyclooxygenase inhibitors. A mitogen-inducible cyclooxygenase gene is thus shown to be regulated by TGF alpha and TPA in rat intestinal epithelial cells. We suggest that products of an intestinal growth factor-inducible cyclooxygenase may play a role in the regulation of mitogenesis.

Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity. An animal model of lipid peroxidation.

These studies examine the in vivo formation of a unique series of PGF2-like compounds (F2-isoprostanes) derived from free radical-catalyzed nonenzymatic peroxidation of arachidonic acid. We have previously shown that levels of these compounds increase up to 50-fold in rats administered CCl4. To understand further the formation of these compounds in vivo, we carried out a series of experiments assessing factors influencing their generation. After CCl4 (2 ml/kg) was administered to rats, plasma F2-isoprostanes increased 55-fold by 4 h. Levels declined thereafter, but at 24 h, they were still elevated 21-fold, indicating continued lipid peroxidation. Pretreatment of rats with isonicotinic acid hydrazide and phenobarbital to induce cytochrome P-450 enhanced the production of F2-isoprostanes after CCl4 administration eightfold and fivefold, respectively, whereas inhibition of the cytochrome P-450 system with SKF-525A and 4-methylpyrazole decreased formation of F2-isoprostanes after CCl4 by 55 and 82%, respectively. Further, the glutathione-depleting agents buthionine sulfoximine and phorone augmented the F2-isoprostane response to CCl4 by 22- and 11-fold, respectively. F2-isoprostanes are formed in situ esterified to lipids and, in addition to increases in levels of free F2-isoprostanes in the circulation, levels of F2-isoprostanes esterified to lipids in various organs and plasma also increase sharply during CCl4 poisoning. The measurement of F2-isoprostanes may facilitate investigation of the role of lipid peroxidation in human diseases.

Prospective multicenter study of eligibility for antiviral therapy among 4,084 U.S. veterans with chronic hepatitis C virus infection

BACKGROUND:Many veterans may not be candidates for hepatitis C virus (HCV) treatment due to contraindications to therapy. The aims of this study were to determine the proportion of HCV-infected veterans who were eligible for interferon alfa and ribavirin therapy and to evaluate barriers to HCV treatment.METHODS:We prospectively enrolled 4,084 veterans who were referred for HCV treatment over a 1-yr period at 24 Veterans Affairs (VA) Medical Centers. Treatment candidacy was assessed using standardized criteria and the opinion of the treating clinician.RESULTS:Overall, 32.2% (95% CI, 30.8–33.7%) were candidates for HCV treatment according to standardized criteria, whereas 40.7% (95% CI, 39.2–42.3%) were candidates in the opinion of the treating clinician. Multivariable analysis identified ongoing substance abuse (OR = 17.68; 95% CI, 12.24–25.53), comorbid medical disease (OR = 9.62; 95% CI, 6.85–13.50), psychiatric disease (OR = 9.45; 95% CI, 6.70–13.32), and advanced liver disease (OR = 8.43; 95% CI, 4.42–16.06) as the strongest predictors of not being a treatment candidate. Among patients who were considered treatment candidates, 76.2% (95% CI, 74.0–78.3%) agreed to be treated and multivariable analysis showed that persons ≥50 yr of age (OR = 1.37; 95% CI, 1.07–1.76) and those with >50 lifetime sexual partners (OR = 1.44; 95% CI, 1.08–1.93) were more likely to decline treatment.CONCLUSIONS:The majority of veteran patients are not suitable candidates for HCV treatment because of substance abuse, psychiatric disease, and comorbid medical disease, and many who are candidates decline therapy. Multidisciplinary collaboration is needed to overcome barriers to HCV therapy in this population.

Effect of oxygen tension on the generation of F2-isoprostanes and malondialdehyde in peroxidizing rat liver microsomes

Although numerous methods have been developed for the detection of lipid peroxidation, it is generally recognized that most of these lack specificity and/or sensitivity, particularly when applied to in vivo situations. We have reported recently that a series of prostaglandin F2-like compounds, termed F2-isoprostanes, are formed in vivo from the free radical catalyzed peroxidation of arachidonic acid and appear to be a useful marker of oxidant stress. Because of formation of other products of lipid peroxidation, such as alkanes and malondialdehyde (MDA), are affected by oxygen tension, which may influence their usefulness as markers of oxidant stress, we carried out a systematic study of the generation of F2-isoprostanes at various oxygen concentrations and compared these changes with the generation of MDA. The disappearance of the F2-isoprostane precursor, arachidonic acid, was used as a reference measure. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N2 and various concentrations of O2 up to 100%. F2-isoprostanes were quantified by mass spectrometry and MDA by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Both MDA and F2-isoprostane formation increased in a time-dependent manner up to 15 min. Their formation correlated with a loss of polyunsaturated fatty acid and with an increase in O2 tension up to 21% O2. At oxygen tensions above 21%, MDA generation continued to increase, while F2-isoprostane generation and arachidonic acid loss did not. Levels of MDA and F2-isoprostanes increased a maximum of 65 and 9.4 times baseline values, respectively. These studies, therefore, define factors that influence the formation of F2-isoprostanes in an in vitro model of lipid peroxidation. Further, they demonstrate that higher O2 tensions do not block formation of F2-isoprostanes and validate their usefulness for assessing lipid peroxidation under high, as well as low, oxygen tension.

THE ISOPROSTANES: UNIQUE PROSTAGLANDIN-LIKE PRODUCTS OF FREE-RADICAL-INITIATED LIPID PEROXIDATION*

The discovery of IsoPs as products of nonenzymatic lipid peroxidation has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology. The quantification of IsoPs as markers of oxidative stress status appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. An important need in the field of free-radical medicine is information regarding the clinical pharmacology of antioxidant agents. Because of the evidence implicating free radicals in the pathogenesis of a number of human diseases, large clinical trials are planned or underway to assess whether antioxidants can either prevent the development or ameliorate the pathology of certain human disorders. However, data regarding the most effective doses and combination of antioxidant agents to use in these clinical trials is lacking. As mentioned previously, administration of antioxidants suppresses the formation of IsoPs, even in normal individuals. Thus, measurement of IsoPs may provide a valuable approach to define the clinical pharmacology of antioxidants. In addition to being markers of oxidative stress, several IsoPs possess potent biological activity. The availability of additional IsoPs in synthetic form should broaden our knowledge concerning the role of these molecules as mediators of oxidant stress. Despite the fact that considerable information has been obtained since the initial report of the discovery of IsoPs [6], much remains to be understood about these molecules. With continued research in this area, we believe that much new information will emerge that will open up additional important new areas for future investigation.

Transforming growth factor alpha protection against drug-induced injury to the rat gastric mucosa in vivo.

This study was designed to determine whether transforming growth factor alpha (TGF alpha) protects rat gastric mucosa against ethanol- and aspirin-induced injury. Systemic administration of TGF alpha dose-dependently decreased 100% ethanol-induced gastric mucosal injury; a dose of 50 micrograms/kg delivered intraperitoneally 15 min before ethanol decreased macroscopic mucosal injury by > 90%. At the microscopic level, TGF alpha prevented deep gastric necrotic lesions and reduced disruption of surface epithelium. Pretreatment with orogastric TGF alpha (200 micrograms/kg) only partially (40%) decreased macroscopic ethanol damage. Intraperitoneal administration of TGF alpha at a dose of 10 micrograms/kg, which does not significantly inhibit gastric acid secretion, decreased aspirin-induced macroscopic damage by > 80%. TGF alpha protection does not seem to be mediated by prostaglandin, glutathione, or ornithine decarboxylase-related events, as evidenced by lack of influence of the inhibition of their production. Pretreatment with the sulfhydryl blocking agent N-ethylmaleimide partially abolished (40%) the protective effect of TGF alpha. In addition, systemic administration of TGF alpha resulted in a two-fold increase in tyrosine phosphorylation of phospholipase C-gamma 1 and in a time- and dose-dependent increase in levels of immunoreactive insoluble gastric mucin; these events occurred in a time frame consistent with their participation in the protective effect of TGF alpha.

ISOPROSTANES—RROSTAGLANDIN-LIKE COMPOUNDS FORMED IN VIVO INDEPENDENTLY OF CYCLOOXYGENASE: Use as Clinical Indicators of Oxidant Damage

F2-isoprostanes are prostanoids produced independently of cyclooxygenase by free radical-catalyzed peroxidation of arachidonic acid-containing lipids. Quantification of F2-isoprostanes from biologic fluids and tissues represents an important advance in the detection and measurement of lipid peroxidation in vivo. In addition, efforts to understand both the biophysical effects of isoprostane containing lipids and the biologic effects of free isoprostanes should lead to a better understanding of the mechanisms responsible for oxidant stress-related alterations in homeostasis. Continued application of F2-isoprostane measurement in experimental models of free radical-induced injury and human disease may allow better design and evaluation of antioxidant therapeutic strategies.

Detection of the major urinary metabolite of prostaglandin D2 in the circulation: Demonstration of elevated levels in patients with disorders of systemic mast cell activation

The symptoms and hemodynamic alterations that accompany episodes of systemic mast cell activation have been largely attributed to excessive prostaglandin (PG)D2 release. Quantification of the major urinary metabolite of PGD2 has been invaluable in elucidating a role for PGD2 in these clinical entities and in the biochemical evaluation of systemic mastocytosis. With the use of a modified mass spectrometric assay for the major urinary metabolite of PGD2, this metabolite was detected in plasma from 10 normal volunteers (3.5 +/- 1.4 pg/ml). Ingestion of niacin, which induces endogenous release of PGD2, increased plasma levels of this metabolite 6.3 to 33 times above the upper limit of normal by 2 hours. Thereafter, levels declined gradually but remained elevated for up to 6 to 8 hours. In contrast, circulating levels of 9 alpha, 11 beta-PGF2, the initial metabolite of PGD2, peaked by 30 minutes and returned to baseline by 2 hours. The clinical utility of measuring the major urinary metabolite in the circulation was demonstrated by detection of markedly increased levels in plasma and serum from patients with systemic mastocytosis and a patient with a severe type I allergic reaction. Thus in the biochemical evaluation of episodes of systemic mast cell activation and endeavors to further elucidate the role of PGD2 in human disease, there are kinetic advantages of measuring the major urinary metabolite of PGD2 in the circulation. One particular advantage is the evaluation of clinical events, which only in retrospect are suspected to be associated with excessive release of PGD2, yet plasma or serum was obtained proximate to the event.

Bromfenac(Duract)-associated hepatic failure requiring liver transplantation

ABSTRACTBromfenac sodium (Duract) is a phenylacetic acid-derived nonsteroidal anti-inflammatory agent introduced in the United States in 1997 and withdrawn in 1998. We describe the first case of fulminant hepatic failure associated with this agent treated successfully with liver transplantation. Similarities to hepatotoxicity with related agents is discussed.

Demonstration of Halothane-induced Hepatic Lipid Peroxidation in Rats by Quantification of Flourine2-Isoprostanes

Background Halothane can be reductively metabolized to free radical intermediates that may initiate lipid peroxidation. Hypoxia and phenobarbital pretreatment in Sprague-Dawley rats increases reductive metabolism of halothane. Flourine2 -isoprostanes, a novel measure of lipid peroxidation in vivo, were used to quantify halothane-induced lipid peroxidation in rats. Methods Rats were exposed to 1% halothane at 21% or 14% Oxygen2 for 2 h. Pretreatments included phenobarbital, isoniazid, or vehicle. Rats also were exposed to halothane, enflurane, and desflurane at 21% Oxygen2. Lipid peroxidation was assessed by mass spectrometric quantification of Flourine2 -isoprostanes. Results Exposure of phenobarbital-pretreated rats to 1% halothane at 21% Oxygen2 for 2 h caused liver and plasma Flourine2 -isoprostane concentrations to increase fivefold compared to nonhalothane control rats. This halothane-induced increase was enhanced by 14% Oxygen sub 2, but hypoxia alone had no significant effect. Alanine aminotransferase activity at 24 h was significantly increased only in the 1% halothane/14% Oxygen2 group. The effect of cytochrome P450 enzyme induction on halothane-induced Flourine2 -isoprostane production and liver injury was determined by comparing the effects of isoniazid and phenobarbital pretreatment with no pretreatment under hypoxic conditions. Halothane caused 4- and 11-fold increases in plasma and liver Flourine2 -isoprostanes, respectively, in non-pretreated rats, whereas isoniazid pretreatment had no effect. Phenobarbital pretreatment potentiated halothane-induced lipid peroxidation with 9- and 20-fold increases in plasma and liver Flourine2 -isoprostanes, respectively. Alanine aminotransferase activity was increased only in this group. At ambient oxygen concentrations, halothane but not enflurane or desflurane, caused Flourine2 -isoprostanes to increase. Conclusions Specific halothane-induced lipid peroxidation was demonstrated in Sprague-Dawley rats using quantification of Flourine2 -isoprostanes and was increased by hypoxia and phenobarbital pretreatment, but not isoniazid pretreatment.