Background: This experimental study examined the effect of the antioxidant drug â€œU-74389Gâ€, on a rat model and particularly in an adrenal ischemia - reperfusion protocol. The effects of that molecule were studied biochemically using blood mean testosterone (T) levels.Materials and methods: 40 rats of mean weight 231.875 g were used in the study. Testosterone levels were measured at 60 min of reperfusion (groups A and C) and at 120 min of reperfusion (groups B and D), A and B without but C and D with U-74389G administration.Results: U-74389G administration significantly increased the T levels by 52.17%+28.69% (p=0.0451). Reperfusion time significantly decreased the T levels by 85.62%+26.33% (P= 0.0019). However, U-74389G administration and reperfusion time together produced a non-significant combined effect in increasing the T levels by 11.18%+17.97% (p= 0.5245).Conclusions: U-74389G administration interacted or not with reperfusion time increased short â€“ term the testosterone levels.Â Â Â Â Â Â Â Â Â

Panoulis, C.

Papalois, A.

Triantafyllou, A.

Tsompos, C.

Î¤Î¿utouzas, K

Î–ografos, G.

ASD Publisher (E-Journals)

Research Journal of Pharmacology and Toxicology 02[01] 2016 www.asdpub.com/index.php/rjpt                                                            ISSN-XXXX-XXXX (Online)                                                  © ASD Publisher All rights reserved.                                                                                                                                                                                                                         1 Original Article  The effect of the antioxidant drug “U-74389G” on testosterone levels during ischemia reperfusion injury in rats  C. Τsompos*1, C. Panoulis2, K Τοutouzas3, A. Triantafyllou4, G. Ζografos5  and A. Papalois6  1Consultant A, Department of Obstetrics & Gynecology, Mesologi County Hospital, Etoloakarnania, Greece 2Assistant Professor, Department of Obstetrics & Gynecology, Aretaieion Hospital, Athens University, Attiki, Greece 3Assistant Professor, Department of Surgery, Ippokrateion General Hospital, Athens University, Attiki, Greece 4Associate Professor, Department of Biologic Chemistry, Athens University, Attiki, Greece 5Professor, Department of Surgery, Ippokrateion General Hospital, Athens University, Attiki, Greece  6Director, Experimental Research Centre ELPEN Pharmaceuticals, S.A. Inc., Co., Pikermi, attiki, Greece   *Corresponding Author C. Τsompos  Consultant A,  Department of Obstetrics & Gynecology, Mesologi County Hospital, Etoloakarnania,  Greece E-mail: Tsomposconstantinos@gmail.com   Keywords: Ischemia,  U-74389G,  Testosterone levels,  Reperfusion    1. Introduction Permanent or transient damage with serious implications on adjacent organs and systems may be due to tissue ischemia - reperfusion (IR). The use of U-74389G in IR has been a challenge for many years. However, although the progress was significant, several practical questions have not clarified. They include: a) how potent U-74389G should be b) when should it be administered and c) at what optimal dose U-74389G should be administered. The promising effect of U-74389G in tissue protection has been noted in several IR studies. U-74389G or also known as 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 which prevents both arachidonic acid-induced and iron-dependent lipid peroxidation[1]. It protects against IR injury in animal organs such as heart, liver and kidney models. These membrane-associating antioxidants are particularly effective in preventing permeability changes in brain microvascular endothelial cells monolayers2. A meta-analysis of 26 published seric variables, coming from the same experimental setting, tried to provide a numeric evaluation of the U-74389G efficacy at the same endpoints (Table 1). Several publications addressed trials of other similar antioxidant molecules to which the studied molecule U-74389G belongs to.  The aim of this experimental study was to examine the effect of the antioxidant drug “U-74389G” on rat model and particularly in a generalized adrenal ischemia - reperfusion (IR) protocol. The effects of that molecule were studied by measuring blood mean testosterone (T) levels.  2. Materials and methods 2.1 Animal preparation This basic experimental research was licensed by Veterinary Address of East Attiki Prefecture under 3693/12-11- 2010 & 14/10-1-2012 decisions. All consumables, equipment and substances, were a grant of Experimental Research Centre of ELPEN Pharmaceuticals Co. Inc. S.A. at Pikermi, Attiki. Accepted standards of human animal care were adopted for Albino female Wistar rats. 7 days pre-experimental normal housing included ad libitum diet in laboratory. Prenarcosis of animals proceeded of continuous intra-experimental general anesthesia3-5, oxygen supply, electrocardiogram and acidometry. Post-experimental euthanasia did not permitted awakening and preservation of the animals. Rats were randomly delivered to four experimental groups by 10 animals in each one, using following protocols of IR: Ischemia for 45 min followed by reperfusion for 60 min (group A); ischemia for 45 min followed by reperfusion for 120 min (group B); ischemia for 45 min followed by immediate U-74389G intravenous (IV) administration and reperfusion for 60 min (group C); ischemia for 45 min followed by immediate U-74389G IV administration and reperfusion for 120 min (group D). The dose of U-74389G was 10 mg/Kg body mass of animals. Ischemia was caused by laparotomic clamping inferior aorta over renal arteries with forceps for 45 min. The clamp removal restored the inferior aorta patency and reperfusion. U-74389G was administered at the time of reperfusion; through catheterized inferior vena cava. The T levels were determined at 60th min of reperfusion (for A and C groups) and at 120th min of reperfusion (for B and D groups). Fourty female Wistar albino rats were used (mean weight 231.875 g [Standard Deviation (SD): 36.59703 g], with minimum weight 165 g and maximum weight 320 g. Rats' weight could be potentially a confusing factor, e.g. more obese rats to have higher T levels. This assumption was also investigated. 2.2 Model of ischemia reperfusion injury Control groups: 20 control rats of mean weight 252.5 g [SD: 39.31988 g] experienced ischemia for 45 min followed by reperfusion.  Group A: Reperfusion which lasted 60 min concerned 10 control rats of mean weight 243 g [SD: 45.77724 g] and mean T levels 0.091 ng/ml [SD: 0.0455705 ng/ml] (Table 2).       Abstract Background: This experimental study examined the effect of the antioxidant drug “U-74389G”, on a rat model and particularly in an adrenal ischemia - reperfusion protocol. The effects of that molecule were studied biochemically using blood mean testosterone (T) levels. Materials and methods: 40 rats of mean weight 231.875 g were used in the study. Testosterone levels were measured at 60 min of reperfusion (groups A and C) and at 120 min of reperfusion (groups B and D), A and B without but C and D with U-74389G administration. Results: U-74389G administration significantly increased the T levels by 52.17%+28.69% (p=0.0451). Reperfusion time significantly decreased the T levels by 85.62%+26.33% (P= 0.0019). However, U-74389G administration and reperfusion time together produced a non-significant combined effect in increasing the T levels by 11.18%+17.97% (p= 0.5245). Conclusions: U-74389G administration interacted or not with reperfusion time increased short – term the testosterone levels.            C. Τsompos et al / The effect of the antioxidant drug “U-74389G” on testosterone levels during ischemia reperfusion injury in rats  © ASD Publisher All rights reserved.                                                                                                                                                                                                                         2 Group B: Reperfusion which lasted 120 min concerned 10 control rats of mean weight 262 g [SD: 31.10913 g] and mean T levels 0.034 ng/ml [SD: 0.0084327 ng/ml] (Table 2).     Lazaroid (L) group: 20 rats of mean weight 211.25 g [SD: 17.53755 g] experienced ischemia for 45 min followed by reperfusion in the beginning of which 10 mg U-74389G /kg body weight were IV administered.  Group C: Reperfusion which lasted 60 min concerned 10 L rats of mean weight 212.5 g [SD: 17.83411 g] and mean T levels 0.137 ng/ml [SD: 0.0851208 ng/ml] (Table 2).     Group D: Reperfusion which lasted 120 min concerned 10 L rats of mean weight 210 g [SD: 18.10463 g] and mean T levels 0.066 ng/ml [SD: 0.0648417 ng/ml] (Table 2).    3. Results  Every weight and T level group was compared with each other by statistical standard t-tests (Table 3). Any significant difference among T levels, was investigated whether owed in any potent significant weight one. The application of generalized linear models (glm) with dependant variable the T levels was followed. The 3 independent variables were the U-74389G or no drug administration, the reperfusion time and both variables in combination. Inserting the rats’ weight also as an independent variable at glm analysis, a non significant relation resulted in (p= 0.1741), so as to further investigation was not needed.  The glm resulted in: U-74389G administration significantly increased the T levels by 0.039 ng/ml [-0.0030339 ng/ml - 0.0810339 ng/ml] (p= 0.0680). This finding was in accordance with the results of standard t-test (p=0.0223). Reperfusion time significantly decreased the T levels by 0.064 ng/ml [-0.1025895 ng/ml - -0.0254105 ng/ml] (p= 0.0018), in accordance also with standard t-test (P= 0.0021). However, U-74389G administration and reperfusion time together produced a non-significant combined effect in increasing the T levels by 0.0083636 ng/ml [-0.0179916 ng/ml - 0.0347188 ng/ml] (p= 0.5245). Reviewing the above and table 3, the tables 4 and 5 sum up concerning the increasing influence of U-74389G along with reperfusion time.   Table 1: The U-74389G influence (+SD) on the levels of some seric variables3 concerning reperfusion (rep) time  Variable 1h rep p-value 1.5h rep p-value 2h rep p-value interaction of U-74389G and rep p-value WBCC +22.99%+12.45% 0.0914 +30.85%+11.14% 0.0045 +38.70%+17.39% 0.0185 +23.45%+6.28% 0.0004 RBCC   +1.39%+0.71% 0.7161 +0.64%+0.32% 0.8106 -0.10%+0.05% 0.9762 +1.05%+0.53% 0.4911 Hematocrit +5.58%+3% 0.0852 +4.73%+2.25% 0.0435 +3.89%+3.44% 0.2608 +3.16%+1.33% 0.0196 Hemoglobin +5.2%+2.8% 0.0925 +3.9%+2.1% 0.0604 +2.7%+3.2% 0.3544 +2.5%+1.3% 0.0423 MCH +1.77%+0.96% 0.0663 +2.40%+0.57% 0.0001 +3.03%+0.71% 0.0003 1.33%+0.36% 0.0005 MCV5 +2.12%+1.16% 0.0663 +2.88%+0.69% 0.0001 +3.64%+0.85% 0.0003 +1.6%+0.43% 0.0005 MCHC -0.5%+0.74% 0.4820 -0.95%+0.63% 0.1124 -1.4%+1.12% 0.1603 -0.69%+0.37% 0.0655 RbcDW -6.13%+3.73% 0.0667 -4.96%+2.27% 0.0175 -3.80%+3.07% 0.1383 -2.54%+1.39% 0.679 Platelet count -17.79%+9.40% 0.0647 -12.83%+5.79% 0.0303 -7.88%+7.83% 0.2939 -6.12%+3.58% 0.0857 Platelet-crit +3.80%+9.87% 0.6373 +9.23%+6.29% 0.1064 +14.66%+9.03% 0.0833 +6.72%+3.73% 0.0712 PDW +1.1%+0.88% 0.2368 +1.79%+0.76% 0.0314 +2.49%+1.33% 0.0807 +0.96%+0.46% 0.0396 Glucose -6.41%+3.50% 0.0663 -8.57%+2.06% 0.0001 -10.74%+2.52% 0.0003 -4.76%+1.28% 0.0005 Creatinine -15.96%+8.71% 0.0663 -21.02%+5.06% 0.0001 -26.09%+6.12% 0.0003 -11.69%+3.16% 0.0005 Uric acid +20.86%+14.44% 0.1614 +15.43%+9.10% 0.0960 +10%+12.11% 0.3946 +4.78%+5.64% 0.3873 Total protein                -5.48%+2.99% 0.0663 -7.34%+1.76% 0.0000 -9.20%+2.16% 0.0000 -4.08%+1.10% 0.0000 γGT +19.35%+18.58% 0.2362 +6.82%+14.89% 0.6442 -5.71%+20.10% 0.7809 +1.23%+9% 0.8877 ALP  +22.66%+12.37% 0.0663 +31.91%+7.69% 0.0001 +41.16%+9.65% 0.0003 +17.75%+4.79% 0.0005 ACP -112.54%+20.95% 0.0006 -128.45%+14.84% 0.0000 -144.36%+21.62% 0.0000 -74.45%+9.63% 0.0000 CPK +54.32%+13.75% 0.0012 +35.34%+17.20% 0.0260 +16.37%+30.24% 0.4951 +18.52%+9.44% 0.0770 LDH +13.56%+7.40% 0.0663 +18.78%+4.52% 0.0001 +24.01%+5.63% 0.0003 +10.43%+2.82% 0.0005 Sodium   +1.22%+0.66% 0.0707 +0.17%+0.61% 0.7714 -0.87%+1.03% 0.3995 -0.32%+0.36% 0.3693 Potassium4 -10.12%+4.82% 0.0579 -2.14%+5.06% 0.6730 +5.83%+6.79% 0.3801 +2.07%+3.03% 0.4853 Chloride   -0.58%+0.77% 0.4533 -0.97%+0.53% 0.0879 -1.36%+0.76% 0.1113 -0.75%+0.38% 0.0159 Calcium    0%+1.75% 1 -0.14%+1.10% 0.8782 -0.28%+1.54% 0.8492 +0.14%+0.64% 0.8245 Phosphorus   -2.23%+5.51% 0.7966 -1.61%+3.32% 0.5789 -1%+4.48% 0.8129 -1.09%+2% 0.5771 Magnesium +1.33%+3.59% 0.7033 -0.28%+2.75% 0.9171 -1.90%+5.28% 0.7161 +0.36%+4.58% 0.8228 Mean -0.01%+27.22% 0.2468 -0.93%+29.14% 0.2265 -1.85%+32.36% 0.2810 -0.40%+16.94% 0.2286   C. Τsompos et al / The effect of the antioxidant drug “U-74389G” on testosterone levels during ischemia reperfusion injury in rats  © ASD Publisher All rights reserved.                                                                                                                                                                                                                         3 Table 2: Weight and testosterone mean levels and Std. Dev. of groups Groups Variable Mean Std. Dev Α Weight 243 g 45.77724 g  Testosterone 0.091 ng/ml 0.0455705 ng/ml B Weight 262 g 31.10913 g  Testosterone 0.034 ng/ml 0.0084327 ng/ml C Weight 212.5 g 17.83411 g  Testosterone 0.137 ng/ml 0.0851208 ng/ml D Weight 210 g 18.10463 g  Testosterone 0.066 ng/ml 0.0648417 ng/ml Table 3: Statistical significance of mean values difference for groups (DG) after statistical standard t test application DG Variable Difference p-value Α-Β Weight -19 g 0.3555  Testosterone 0.057 ng/ml 0.0031 Α-C                    Weight 30.5 g 0.0674  Testosterone -0.046 ng/ml 0.1030 Α-D                    Weight 33 g 0.0574  Testosterone 0.025 ng/ml 0.2674 Β-C Weight 49.5 g 0.0062  Testosterone -0.103 ng/ml 0.0043 Β-D                    Weight 52 g 0.0009  Testosterone -0.032 ng/ml 0.1369 C-D Weight 2.5 g 0.7043  Testosterone 0.071 ng/ml 0.0662  Table 4: The increasing influence of U-74389G in connection with reperfusion time Increase 95% c. in Reperfusion time p-values    t-test glm 0.046 ng/ml -0.018146 ng/ml -0.110146 ng/ml 1h 0.1030 0.1493 0.039 ng/ml -0.0030339 ng/ml - 0.0810339ng/ml 1.5h 0.0223 0.0680 0.032 ng/ml -0.0114416 ng/ml - 0.0754416ng/ml - 2h 0.1369 0.1391 0.064 ng/ml -0.1025895 ng/ml -0.0254105 ng/ml reperfusion time 0.0021 0.0018 0.0083636 ng/ml -0.0179916 ng/ml - 0.0347188ng/ml interaction - 0.5245   Table 5: The (%) increasing influence of U-74389G in connection with reperfusion time Increase +SD Reperfusion time p-values 40.35% +28.70% 1h 0.1261 52.17% +28.69% 1.5h 0.0451 64% +44.32% 2h 0.1380 -85.62% +26.33% reperfusion time 0.0019 11.18% +17.97% interaction 0.5245  4. Discussion T is considered a reliable index substance of adrenals metabolism; being of great clinical significance. Examples are described herein concerning whether adrenal ischemia can influence the T levels. Cakir E et al found [6] total T levels significantly higher in PCOS women also having higher risk for cardiovascular disease (CVD) and myocardial ischemia than control subjects. Guven S et al found [7] significantly higher concentrations of serum total T (P = 0.031), elevated serum ischemia-modified albumin (IMA) concentrations - a clinical marker of ongoing myocardial ischemia - well correlated with total T levels (P = 0.022) in women with PCOS than control group. Shaw LJ et al [8] found more frequent and heavy angiographic coronary artery disease (CAD) (P = 0.04) and 9.8% less cumulative 5-yr cardiovascular (CV) event-free survival (P = 0.006) in women with clinical features of PCOS as defined by top T quartile (> 30.9 ng/dl) than normal control women. PCOS remained a significant predictor (P < 0.01) in prognostic models for suspected ischemia and CV disease. Kovalenko AN et al [9] determined peripheral blood T concentrations with consequences on metabolic background conducting to cerebral atherosclerosis and ischemic stroke development in clinically normal elderly than younger subjects.  5. Conclusion U-74389G administration interacted or not with reperfusion time increased short – term the testosterone levels. The intervention of this molecule into the biosynthetic pathway of T worths further investigation.        Acknowledgment: This study was funded by Scholarship by the Experimental Research Center ELPEN Pharmaceuticals (E.R.C.E), Athens, Greece. The research facilities for this project were provided by the aforementioned institution.  References [1] https://www.caymanchem.com/app/template/Product.vm/catalog/75860. [2] Fenglin Shi, Jennifer Cavitt, Kenneth L Audus. 21-aminosteroid and 2-(aminomethyl)chromans inhibition of arachidonic acid-induced lipid peroxidation and permeability enhancement in bovine brain microvessel endothelial cell monolayers. Free Radical Biology and Medicine, 1995; 19(3): 349-357. [3] Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Ζografos G, et al. The Effect of the Antioxidant Drug “U-74389G” on Lactate Dehydrogenase Levels during Ischemia Reperfusion Injury in Rats. J Anal Pharm Res 2016; 3(1): 00043. [4] Τsompos C., Panoulis C., Τοutouzas K., Ζografos G., Papalois A. The trends of the antioxidant drug “U-74389G” on potassium levels during hypoxia reoxygenation injury in rats. Periodicum Biologorum 2016; 118(2): 105–110. [5] Τsompos C, Panoulis C, Τοutouzas K, Triantafyllou A, Ζografos G, et al. The Acute Effect of the Antioxidant Drug “U-74389G” on Mean Corpuscular Volume Levels during Ischemia Reperfusion Injury in Rats. Research Journal of Pharmacology and Toxicology 2015; 01[05]: 54-57. [6] Cakir E, Ozbek M, Sahin M, et al: Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients. J Ovarian Res. 2012 Dec 18; 5(1):45.  [7] Guven S, Karahan SC, Bayram C, et al: Elevated concentrations of serum ischaemia-modified albumin in PCOS, a novel ischaemia marker of coronary artery disease. Reprod Biomed Online. 2009 Oct; 19(4):493-500. [8] Shaw LJ, Bairey Merz CN, Azziz R, et al: Postmenopausal women with a history of irregular menses and elevated androgen measurements at high risk for worsening cardiovascular event-free survival: results from the National Institutes of Health--National Heart, Lung, and Blood Institute sponsored Women's Ischemia Syndrome Evaluation. J Clin Endocrinol Metab. 2008 Apr; 93(4):1276-84.  [9] Kovalenko AN, Kostiuchenko VG. Gonadal and gonadotropic function of late middle-aged men and women with sequelae of ischemic stroke. Zh Nevropatol Psikhiatr Im S S Korsakova. 1987; 87(1):29-33. 

The effect of the antioxidant drug â€œU-74389Gâ€ on testosterone levels during ischemia reperfusion injury in rats

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The effect of the antioxidant drug â€œU-74389Gâ€ on testosterone levels during ischemia reperfusion injury in rats

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