Lazaroid U-74389G Research Articles

Decrease in γ-Glutamyltransferase Activity in Rat Type II Cells Exposed in Vitro to Hyperoxia: Effects of the 21-Aminosteroid U-74389G

Although the effect of hyperoxia on antioxidant enzymes is well known, the effect of subtoxic levels of hyperoxia on γ-glutamyltransferase (γ-GT), involved in the degradation and uptake of extracellular GSH for intracellular GSH synthesis, is unknown. The aim of the study was to investigate (1) the effects of in vitro hyperoxia on γ-GT activity of type II cells and (2) the effects of the lazaroid U-74389G and N-acetylcysteine (NAC) on the hyperoxia-induced changes in γ-GT and antioxidant enzyme activities. At 48 h after isolation, rat type II cells were exposed for 2 days to air, 60% O2 or 85% O2, with or without 30 μM U-74389G or 100 μM NAC. After the exposure, the cells were harvested and assayed for superoxide dismutase (SOD), glutathione peroxidase (GPx), γ-GT activity, and GSH levels. In another series of experiments 85% O2-exposed cells, with or without U-74389G, were used for Northern blotting of γ-GT mRNA. Exposure to 60% O2 decreased γ-GT and GSH by -47 and -34%, respectively, while SOD and GPx activities remained unchanged. After 85% O2-exposure γ-GT decreased by -55%, SOD and GPx increased by +55 and +87%, respectively, while GSH decreased by -35%. NAC treatment decreased γ-GT activity by -42% in the air-exposed cells. After 60% O2, U-74389G led to significantly higher γ-GT (+117%,) and GSH (+26%) while NAC only led to higher GSH (+28%) compared to the oxygen-exposed cells not treated with NAC or U-74389G. After 85% O2, U-74389G increased γ-GT, SOD, and GSH by +72, +58, and +68%, respectively, while NAC only increased SOD (+49%) and GSH (+26%) compared to the oxygen-exposed cells not treated with NAC or U-74389G. The 85%, O2 exposure, with or without U-74389G, had no effect on γ-GT mRNA levels. The results show that hyperoxia decreases rat type II cell γ-GT activity in vitro. This effect was not related to an altered regulation at mRNA level and it was not associated with the hyperoxia-induced decrease in intracellular GSH, since restoration of the GSH levels by NAC did not restore γ-GT activity. The lazaroid U-74389G with vitamin E-like properties effectively prevented the decrease in γ-GT and GSH, so that direct inactivation of the membrane-bound γ-GT by hyperoxia is the most likely mechanism.

Variable efficacy of radical scavengers and iron chelators to attenuate gentamicin ototoxicity in guinea pig in vivo

Recent studies from our laboratory have suggested that the ototoxic side effects of gentamicin are caused by a metabolized or ‘activated’ form of the drug. Furthermore, we have postulated that the activation proceeds via the formation of an iron-gentamicin complex and that this complex produces free radicals. The present study assessed the protective effects of free radical scavengers and iron chelators on gentamicin-induced ototoxicity in guinea pigs in vivo. Gentamicin (120 mg/kg per day for 19 days) caused progressive threshold shifts reaching 50–65 dB at 18 kHz. Co-therapy with different radical scavengers yielded results ranging from no protection (with allopurinol, dimethyl sulfoxide, benzoate, lazaroid U74389G) to a moderate attenuation of hearing loss (with mannitol, 4-methylthiobenzoate, WR-2721). This finding agrees well with previous reports of inconsistent effects of scavengers on aminoglycoside-induced ototoxicity although it should be cautioned that only a single dose and route of application was tested. Two iron chelators, deferoxamine and 2,3-dihydroxybenzoate, significantly reduced the gentamicin-induced threshold shifts to about 10 dB or less. Iron chelators markedly decreased total serum iron levels while gentamicin treatment alone had no influence. There were no differences in serum gentamicin levels among all treated groups. This study confirms that iron plays a critical role in gentamicin ototoxicity and suggests that iron chelators, which are well-established drugs in clinical therapy, may be promising therapeutic agents to reduce aminoglycoside ototoxicity.

Cardioprotective effects of the lazaroid U74389G following cold preservation and transplantation of rat hearts.

Limited recovery of contractile function and loss of coronary reactivity have been observed following prolonged hypothermic storage and transplantation of the heart. Since lipid peroxidation has a significant role in these deficits, we investigated the cardioprotective effects of a 21-aminosteroid. U74389G, 3 mg/kg i.v., was given daily for 2 consecutive days to donor Lewis rats before the hearts were harvested and to recipient Lewis rats for 3 consecutive days after heart transplantation. Donor hearts were preserved for 4 hr in cold saline (4 degree C) before transplantation. Left ventricular developed pressure (LVP), basal coronary perfusion, and coronary reactivity to endothelium-dependent dilation (bradykinin, 0.1 microM) or endothelium-dependent dilation (sodium nitroprusside, 0.5 microM) were studied in isolated, buffer-perfused heart, using a modified Langendorff model. Cold preservation alone significantly reduced LVP and coronary perfusion. Coronary reactivity to bradykinin and sodium nitroprusside was also significantly impaired. In U74389G-treated donor hearts, 4 hr of cold ischemia did not alter contractile function, coronary perfusion or endothelial reactivity, although the response to sodium nitroprusside did not fully recover. In untreated recipients, in vivo reperfusion (transplantation) resulted in reduced LVP and perfusion deficits. Treating donors and recipients with U74389G improved left ventricular contractibility and coronary perfusion, although endothelium-dependent and -independent coronary reactivity remained affected. These results indicate that the lazaroid U74389G exerts significant cardioprotection during both preservation and transplantation of the heart. Donor and recipient pretreatment is mandatory for maximal efficacy with U74389G.

The lazaroid U74389G protects normal brain from stereotactic radiosurgery-induced radiation injury

Purpose : To test an established model of stereotactic radiosurgery-induced radiation injury with pretreatments of either methylprednisolone or the lazaroid U74389G. Methods and Materials : Nine cats received stereotactic radiosurgery with a linear accelerator using an animal radiosurgery device. Each received a dose of 125.0 Gy prescribed to the 84% isodose shell to the anterior limb of the right internal capsule. One animal received no pretreatment, two received citrate vehicle, three received 30 mg/kg of methylprednisolone, and three received 5 mg/kg of U74389G. After irradiation, the animals had frequent neurologic examinations, and neurologic deficits developed in all of them. Six months after the radiation treatment, the animals were anesthetized, and had gadolinium-enhanced magnetic resonance (MR) scans, followed by Evans blue dye perfusion, euthanasia, and brain fixation. Results : Magnetic resonance scans revealed a decrease in the size of the lesions from a mean volume of 0.45 ± 0.06 cm 3 in the control, vehicle-treated, and methylprednisolone-treated animals to 0.22 ± 0.14 cm 3 in the U74389G-treated group. The scans also suggested the absence of necrosis and ventricular dilatation in the lazaroid-treated group. Gross pathology revealed that lesions produced in the untreated, vehicle-treated, and methylprednisolone-treated cats were similar and were characterized by a peripheral zone of Evans blue dye staining with a central zone of a mature coagulative necrosis and focal hemorrhage. However, in the U74389G-treated animals, the lesions were found to have an area of Evans blue dye staining, but lacked discrete areas of necrosis and hemorrhage. Conclusion : These results suggest that the lazaroid U74389G protects the normal brain from radiation injury produced by stereotactic radiosurgery.

Lazaroid U-74389G for 48-hour canine liver preservation.

Lazaroids have been reported to attenuate preservation and reperfusion injury. In this study, we examined whether lazaroids can improve the outcome after 48-hr canine liver preservation and transplantation. Adult female beagle dogs were randomized into 4 dosage groups (5 animals each). Lazaroid U-74389G was intravenously administered at a dose of 0 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg to donors 30 min before harvesting and also to recipients 30 min before revascularization. Control animals (0 mg/kg) were given the lazaroid vehicle. The liver grafts were orthotopically transplanted after 48 hr of hypothermic preservation in UW solution. Lazaroid treatment significantly improved outcome after transplantation. Five-day animal survival increased from 0% in the control to 60% in the 6 mg/kg group, 100% in the 10 mg/kg group, and 80% in the 15 mg/kg group. Lazaroid protected the hepatocytes from damage during preservation, and enhanced energy charge and hepatic blood flow after reperfusion. Histological alterations were significantly less severe in the lazaroid-treated groups. The area of necrotic hepatocytes decreased from 43.7 +/- 17.7 in the control to 13.5 +/- 3.0 in the lazaroid 10 mg/kg group. These results indicate that lazaroid U-74389G has potential for improvement of clinical liver preservation.

Hydrogen peroxide induces 21-aminosteroid-inhibitable F2-isoprostane production and cytolysis in renal tubular epithelial cells.

F2-isoprostanes are the newly identified reactive oxygen species-catalyzed peroxidation products of arachidonate. The infusion of these prostaglandin F2-like prostanaoids into the rat kidney induces profound parallel reductions in RBF and GFR, suggesting that these metabolites may be partly responsible for the hemodynamic alterations seen in free radical-linked acute renal injury models. The present study examined directly in renal proximal tubular (LLC-PK1) cells whether hydrogen peroxide, a reactive oxygen species implicated in many models of acute renal injury, induces F2-isoprostane production and whether its production can be inhibited by the recently synthesized lipid peroxidation inhibitor 21-aminosteroid (lazaroid U-74389G). The incubation of LLC-PK1 cell layers with hydrogen peroxide for 3 h resulted in a dose-related six-fold increase in F2-isoprostane production, measured by the gas chromatographic-mass spectroscopic method. The preincubation of cells with 21-aminosteroid prevented hydrogen peroxide-induced F2-isoprostane production, a finding also demonstrable with other lipid peroxidation inhibitors, e.g., 2-methyl aminochroman (U-83836E) and diphenyl-p-phenylenediamine. Besides inhibiting isoprostane production, 21-aminosteroid reduced hydrogen peroxide-induced lipid degradation and peroxidation, and protected the cells against hydrogen peroxide-induced cytolysis. The novel finding that hydrogen peroxide induces 21-aminosteroid-inhibitable F2-isoprostane production in renal epithelial cells supports the in vivo report that its levels are elevated in reactive oxygen species-linked renal injury models such as ischemia-reperfusion. Besides direct cell injury, lipid peroxidation by generating F2-isoprostanes may further contribute to renal dysfunction through a vasoconstrictive mechanism. Thus, the inhibition of excess F2-isoprostane production may be one of the additional mechanisms, besides cytoprotection, by which antioxidants ameliorate renal dysfunction in experimental models of acute renal injury.

Improved small intestinal preservation after lazaroid U74389G treatment and cold storage in University of Wisconsin solution.

The small intestine (SI) is highly sensitive to oxygen free radical-induced injury. The most common preservation solution, University of Wisconsin (UW) solution, does not adequately prevent free radical-induced injury. Lazaroids, and U74389G in particular, are a new class of compound that are potent inhibitors of superoxide-mediated lipid peroxidation. We studied the added influence of U74389G to 18-hr cold preservation of rat SI in UW solution. Three groups of rats were studied. In group 1, SI was excised and reperfused immediately. In group 2, SI was stored in UW solution at 4 degrees C for 18 hr. In group 3, U74389G was given to the SI graft before storage and again before reperfusion. Blood reperfusion of the grafts was achieved via connection to the superior mesenteric artery and portal vein of support rats. Functional recovery was assessed using a maltose tolerance test. Weight changes were calculated and histologic studies done. After 30 and 60 min of reperfusion, maltose uptake in group 3 was significantly better than that of the group 2, and returned to control levels. Significantly more tissue swelling was noted in group 3 over control, but the magnitude was less than that of group 2. Less transmural necrosis and villous blunting were noted in group 3 versus group 2; the appearance of the mucosa in group 3 approached that of group 1. We conclude that the use of U74389G treatment in addition to cold storage in UW solution improves recovery of graft function and minimizes morphologic damage to the small intestinal mucosa.