The Effect of the Antioxidant Drug U-74389G on Magnesium Levels During Hypoxia–Reoxygenation Injury in Rats

Hypoxia–reoxygenation (HR) is recognized for specific events that occur following an injury and common signs and symptoms associated with tissue damage and repair [1]. A critical step in this context is the deepening of the understanding of how the body repairs damaged tissues using antioxidant substances. However, although the progress was significant, several practical questions have not been clarified. They include a) how potent the antioxidant should be, b) when should it be administered, and c) at which optimal DOI: 10.15690/vramn.v70.i4.1405 C. Τsompos1, C. Panoulis2, K. Τοutouzas3, G. Ζografos3, A. Papalois4 1 Mesologi County Hospital, Etoloakarnania, Greece 2 Aretaieion Hospital, Athens University, Attiki, Greece 3 Ippokrateion General Hospital, Athens University, Attiki, Greece 4 Exprerimental Research Center ELPEN Pharmaceuticals, S.A. Inc., Co.


INTRODUCTION
dose the drug should be administered. The promising effect of the antioxidant, the aminosteroid U-74389G in tissue protection has been noted in several studies. International bibliography demonstrated that U-74389G which belongs to the family of aminosteroids (lazaroids), is very popular in HR experiments (Table 1). U-74389G (or 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-pregna-1,4,9(11)triene-3,20-dione maleate salt) is an antioxidant factor [2]. Its main capacity is both arachidonic acid-induced and irondependent lipid peroxidation prevention. It has been proved beneficial in some animal tissues as heart, liver and kidney ischemia-reperfusion (IR) models, as well in protecting brain microvascular endothelial cells monolayers against permeability changes [3]. The aim of this experimental study was to examine the effect of the antioxidant drug U-74389G in a rat model of HR using blood magnesium (Mg 2+ ) levels as possible biomarker of effect of the drug.

Study design
A controlled study was performed.

Eligibility criteria
Forty female Wistar albino rats (mean weight 231.9±36.9 g) were used.

Testing environment
This experimental study was performed at the Exprerimental Research Center of ELPEN Pharmaceuticals Co. Inc. S.A. at Pikermi, Attiki (Greece). All settings needed for the study including consumables, equipment and substances used, were a courtesy of that S.A.

Duration of study
The experiment lasted 15 days.

Description of medical intervention
The experiment started with prenarcosis followed by general anesthesia of each animal. The detailed anesthesiologic technique was described in related references [4,5]. Continuous oxygen supply was administered during whole experiment performance. The electrocardiogram and acidometry were continuously monitored. Hypoxia was caused by clamping inferior aorta over renal arteries with forceps for 45 min after laparotomic access was achieved. Reoxygenation was induced by removing the clamp and reestablishment of inferior aorta patency. After exclusion of a blood flow, the protocol of HR was applied as described above for each of experimental group. U-74389G was administered at the time of reoxygenation through inferior vena cava after the catheterization had been achieved.

Study outcomes
Primary outcome of the study Control groups included 20 control rats (252.5±39.3 g) submitted to hypoxia for 45 min followed by reoxygenation.

Subgroup analysis
Rats were randomly assigned to four experimental groups (10 animals in each group) using following protocols of HR: hypoxia for 45 min followed by reoxygenation for 60 min (group A); hypoxia for 45 min followed by reoxygenation for 120 min (group B); hypoxia for 45 min followed by immediate intravenous administration of U-74389G and reoxygenation for 60 min (group C); hypoxia for 45 min followed by immediate intravenous administration of U-74389G and reoxygenation for 120 min (group D). The dose of U-74389G was 10 mg/Kg body mass of animals.

Outcome recording techniques
The Mg 2+ levels were determined at 60 th min of reoxygenation (groups A and C) and at 120 th min of reoxygenation (groups B and D).

Ethics review
The study was licensed by Veterinary Address of East Attiki Prefecture under 3693/12-11-2010 & 14/10-1-2012 decisions. Animals were used and maintained in accordance with accepted standards of humane animal care.

Statistical processing of data
Rats of each group were compared by weight and level of Mg 2+ in blood with each one by statistical paired t-test ( Table  3). The application of generalized linear models (glm) with dependant variables (Mg 2+ levels) and independent variables U-74389G or no drug, the reoxygenation time and both variables in combination was performed. The data for the dependant variables (Mg 2+ levels) are depicted at Table 2. The dummy variables for U-74389G or no drug were stand for 1 or 0 respectively. The dummy variables for reoxygenation time were stand as 1 and 2 for 60 min and 120 min respectively. The dummy variables for their combination were found by their numerical

Study object
The study was carried out on forty (40) 16-18 week-old female albino rats of Wistar strain having minimum weight 165 g and maximum weight 320 g (mean weight 231.9±36.9 g). The animals were divided into 4 groups.

Key fi ndings
U-74389G administration possessed a trend to decrease the Mg 2+ levels by 0.01 mg/dl (-0.20-0.18 mg/dl; p =0.916). This finding was in accordance with the results of a paired t-test (p =0.919). Reoxygenation time non-significantly increased the Mg 2+ levels by 0.15 mg/dl (-0.03-0.33 mg/dl; p =0.106), also in accordance with the paired t-test results (p =0.109). However, U-74389G administration and reoxygenation time in combination non-significantly increased Mg 2+ levels by 0.01 mg/dl (-0.10-0.13 mg/dl; p =0.823). Reviewing the above and Table 3, the Table 4 sums up the effect of U-74389G regarding reoxygenation time. Considering the weight of animals as an independent variable in generalized linear models analysis, a non-significant association with Mg 2+ levels was revealed (p =0.495), suggesting that further investigations were not needed.

DISCUSSION
Unpleasantly, studies describing whether hypoxia may affect the Mg 2+ levels have not been found in published literature. However, there were a lot of studies reporting how the fluctuations of Mg 2+ levels affect functions of various organs. Nevertheless, isolated magnesium administration is impossible. However, it is meant that another drug or ion administered with Mg 2+ might affect the Mg 2+ levels. Siegler J.E. et al. found [6] non-significant difference in worse outcome (neurologic deterioration, ND), death, discharge disposition or poor short-term functional measures) in ischemic stroke patients, whose serum Mg 2+ levels were decreased from a baseline during the first 24 hours of admission compared to patients with unchanged or increasing Mg 2+ levels. Lee K.C. et al. considered [7] as safe the combination of St. Thomas cardioplegic arrest with histidine-tryptophan-ketoglutarate solution with an average short-term ischemia survival time ~225 minutes, nearly similar to other approaches, during lowpressure perfusion donor heart preservation. van den Bergh W.M. et al. tried [8] to prevent or reverse delayed cerebral ischemia (DCI) after aneurysmal subarachnoid haemorrhage (SAH) by administering magnesium as a neuroprotective agent at a continuous intravenous dosage of 64 mmol/L per day, which maintained serum Mg 2+ levels within the range of 1.0-2.0 mmol/L for 14 days. Whitelaw A. et al. treated [9] hypoxic-ischemic labor-delivery-encephalopathy infants with magnesium sulphate. Ichiba H. et al. found [10] significantly   [15] learning ability, injecting either 0.3 μg or 0.06 μg intrastriatally ferric chloride (FeCl 3 ), one week after a 60-minute oligemic IR median brain episode in adult rats. Lazaroid U-74389G, a potent inhibitor of iron-induced lipid peroxidation, totally prevents the learning impairments in both median adult and aged animals, suggesting that iron-induced lipid peroxidation may be responsible for the late learning deficiencies. However, when U-74389G was administered alone one week after the oligemic episode, it also impaired the animals' learning ability. Moore R.M. et al. subjected [16] horses into IR condition with a reduced to 20% of baseline (BL) colonic arterial blood flow for 3 hours. Then the colon was reperfused during another 3 hours. 21-Aminosteroid U-74389G administered as 10 mg/kg intravenously 30 minutes prior to colonic reperfusion significantly increased mean pulmonary artery and mean right atrial pressure and decreased colonic arterial resistance at least for 3 hours during IR. These data demonstrate the potential of the 21-aminosteroid U-74389G to affect reperfusion, however, the concentration of Mg 2+ in blood serum seem to be a pure biomarker of the effect of the drug in HR.
It is repeated that it is unknown how hypoxia affects the serum Mg 2+ levels. Nevertheless, Mg 2+ intravenous infusion has beneficial protective effects for nervous and cardiac muscle tissues and for respiratory acidosis [9,10]. U-74389G although reinforces reperfusion does not protects nervous tissue if the criterion is the leaning ability and of course has unknown effects on serum Mg 2+ levels. However this study although tried to correlate directly the effect of U-74389G on the serum Mg 2+ levels, the outcome was non significant and confusing.

CONCLUSION
Administration of U-74389G, reoxygenation time and their interaction provide only a statistically non-significant trend for a short-term effects in Mg 2+ levels. Levels of Mg 2+ cannot be considered as biomarkers of the effect of U-74389G at least at studied dose. Study suggests that the longer study time or increased U-74389G dose may reveal any significant effects post-HR.