Fevzi Nuri Aydin

Effects of various cryoprotectants on bull sperm quality, DNA integrity and oxidative stress parameters ☆

The objectives of this study was to compare the effects of type and concentration of cryoprotectants glycerol (G), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the plasma membrane and DNA integrity as well as antioxidant activity of cryopreserved Eastern Anatolian red bull sperm. Ejaculates were collected from the three bulls using an artificial vagina twice a week. The ejaculates were pooled to increase the semen volume for replication and to eliminate variability among the evaluated samples. The pooled ejaculates were also split into seven equal experimental groups and diluted with the modified base extender to a final spermatozoa concentration of 15×10(6)/ml. The extended samples were cooled slowly to 4°C and equilibrated for 4h. They were then loaded into 0.25ml French straws and frozen using a digital freezing machine at 3 programmed rates: -3°C/min from +4°C to -10°C, -40°C/min from -10°C to -100°C, and -20°C/min from -100°C to -140°C. Thereafter, the straws were plunged into liquid nitrogen at -196°C. Frozen straws were thawed individually at 37°C for 30s in a water bath to analyse progressive motility and sperm motion characteristics as well as membrane integrity using hypo-osmotic swelling test. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay using Image Analysis System. 6% G exhibited the greatest percentages of CASA (43.7±2.92%) and progressive (26.4±2.64%) motilities when compared to the other groups (P<0.001). 6% G and 6% EG showed the greatest values of preserved membrane integrity (P<0.001). 6% DMSO and 3% EG + 3% DMSO resulted in greater chromatin damage than the other groups (P<0.001). The antioxidant activities of GPx, GSH, and CAT as well as the total antioxidant activity were affected by the type of cryoprotectant; notably, 2% G+2% EG+2% DMSO yielded the lowest activities when compared to the other groups (P<0.001). In conclusion, no advantages were found in using EG or DMSO to replace G in bull sperm cryopreservation. Freezing with cryoprotectant 6% G yielded the best post-thaw sperm characteristics for Eastern Anatolian Red bull spermatozoa.

The relation between oxidative stress, inflammation, and neopterin in the paraquat-induced lung toxicity

Paraquat (PQ) is a well-known quaternary nitrogen herbicide. The major target organ in PQ poisoning is the lung. Reactive oxygen species (ROS) and inflammation play a crucial role in the development of PQ-induced pulmonary injury. Neopterin is synthesized in macrophage by interferon γ and other cytokines. We aimed to evaluate the utility of neopterin as a diagnostic marker in PQ-induced lung toxicity. Sprague Dawley rats were randomly divided into two groups (sham and PQ), administered intraperitoneally 1 mL saline and PQ (15 mg/kg/mL) respectively. Blood samples and lungs were collected for analyses. Lung injury and fibrosis were seen in the PQ group. Serum total antioxidant capacity, lactate dehydrogenase (LDH), and lung transforming growth factor-1β (TGF-1β) levels were significantly higher than the sham group (in all, p < 0.001). In addition, in the PQ group, serum neopterin and lung malondialdehyde (MDA) levels were also significantly higher than the sham group (in all, p = 0.001). Serum neopterin levels were correlated with LDH activities, lung MDA, lung TGF-1β levels, and the degree of lung injury. These findings demonstrated that oxidative stress, reduction of antioxidant capacity, and inflammation play a crucial role in the PQ-induced lung injury. Elevated serum neopterin levels may be a prognostic parameter to determine extends of PQ-induced lung toxicity. Further studies may be performed to clarify the role of neopterin by different doses of PQ.

Pentraxin 3 as a potential biomarker of acetaminophen-induced liver injury.

OBJECTIVE Overdose of acetaminophen (APAP) can lead to severe liver injury in humans and experimental animals. Pentraxin-3 (PTX-3) is produced and released by several cell types. In this study, we aimed to evaluate whether PTX-3 is a potential biomarker in the identification of APAP-induced liver injury. MATERIALS AND METHODS Thirty adult male Wistar rats were randomly divided into three groups: control, APAP-1 and APAP-2 groups. APAP-1 (1 g/kg) and APAP-2 (2 g/kg) group rats were given APAP by gastric tube. Liver tissues and blood samples were obtained for biochemical and histopathological analysis. Biochemical parameters, plasma and liver PTX-3 levels and degree of liver necrosis were measured in all groups. RESULTS APAP treatments caused necrosis in liver and accompanied by elevated liver PTX-3 levels after 48 h. In APAP-1 and APAP-2 groups when compared with control group (7.5±3.3 ng/mg protein), mean liver PTX-3 concentrations were 14.1±3.0 (p=0.032) and 28.5±8.2 (p<0.001) ng/mg protein, respectively. All rats (100%) in the APAP-2 group had the degree 3 liver necrosis. However 10%, 40% and 50% of rats had the degree 1, the degree 2 and the degree 3 liver necrosis in the APAP-1 group, respectively. The degrees of liver necrosis of the APAP-1 and APAP-2 groups were higher than the group of control (p<0.001 and p<0.001, respectively). CONCLUSIONS PTX-3 may have a role in the APAP-induced liver injury in the rats. The elevated liver PTX-3 in the APAP-induced hepatic necrosis might be a marker of acute histological liver damage. Further prospective studies are necessary to clarify the prognostic value of liver PTX-3 for prediction of histological hepatic necrosis in the APAP-induced liver injury.

The association between neopterin and acetaminophen-induced nephrotoxicity

Introduction: In large dosages, acetaminophen (APAP) produces acute kidney necrosis in most mammalian species. High neopterin levels have been accepted as strong indicators for the clinical severity of some diseases. In this study, we aimed to evaluate whether neopterin is a biomarker in the identification of APAP-induced nephrotoxicity. Materials and Methods: Thirty adult male Wistar rats were randomly divided into three groups: control, APAP-1, and APAP-2 groups. APAP-1 and APAP-2 group rats were given a single dose of 1 and 2 g/kg body weight of APAP by gastric tube, respectively. Kidney tissues and blood samples were obtained for biochemical and histopathological analyses. Biochemical parameters, serum and kidney neopterin levels, and the grade of tubular injury were compared in the control, APAP-1, and APAP-2 group animals. Results: APAP treatments caused tubular necrosis in the kidney and increase in serum creatinine concentrations accompanied by elevated serum and kidney neopterin levels. In the rats of groups APAP-1 and APAP-2 when compared with that of the control group (109.1 pmol/mg protein), median kidney neopterin concentrations were 162.1 (p = 0.089) and 222.2 (p < 0.001) pmol/mg protein, respectively. The grade of tubular injury of the APAP-1 and APAP-2 groups was higher than the group of control (both p < 0.001). Conclusions: Serum and kidney neopterin levels could be sensible alternative to evaluate the risk to have nephrotoxicity because of APAP overdose. The elevated serum and kidney neopterin in the APAP-induced tubular necrosis might be a marker of acute histological kidney injury.

The Effects of N-Acetylcysteine and Ozone Therapy on Oxidative Stress and Inflammation in Acetaminophen-Induced Nephrotoxicity Model

Introduction: Acetaminophen (APAP) is an analgesic and antipyretic agent. In overdoses, it is associated with nephrotoxicity. We examined the potential protective effects of N-acetylcysteine (NAC) and NAC + ozone therapy (OT) combination against APAP-induced nephrotoxicity. Materials and methods: Thirty-two male Sprague–Dawley rats were divided into four groups: sham, control (APAP), NAC, and NAC + OT. In the APAP, NAC, and NAC + OT groups, kidney injury was induced by oral administration of 1 g/kg APAP. The NAC group received NAC (100 mg/kg/day). NAC + OT group received NAC (100 mg/kg/day) and ozone/oxygen mixture (0.7 mg/kg/day) intraperitoneally for 5 days immediately after APAP administration. All animals were killed at 5 days after APAP administration. Renal tissues and blood samples were obtained for biochemical and histopathological analyses. Neopterin, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-10 levels were measured in sera. Malondialdehyde (MDA) levels and glutathione peroxidase (GPx) activities were determined in renal homogenates. Results: NAC and NAC + OT significantly decreased MDA and TNF-α levels and increased IL-10 levels and GPx activities. Serum neopterin and IL-6 levels were not different among all groups. APAP administration caused tubular necrosis in the kidney. The degrees of renal necrosis of the APAP group were higher than the other groups. Renal injury in rats treated with combination of NAC and OT were found to be significantly less than the other groups. Conclusions: Our results showed that NAC and OT prevented renal injury in rats and reduced inflammation. These findings suggest that combination of NAC and OT might improve renal damages because of both oxidative stress and inflammation.

Hyperbaric oxygen treatment and N-acetylcysteine ameliorate acetaminophen-induced liver injury in a rat model.

An overdose of acetaminophen (APAP) produces centrilobular hepatocellular necrosis. We aimed to investigate the hepatoprotective effects of N-acetylcysteine (NAC) only and hyperbaric oxygen (O2) treatment (HBOT) combined with NAC, and their anti-inflammatory properties in liver tissue. In the current study, a total of 32 male Sprague Dawley rats were divided into 4 groups: sham, APAP, NAC, and NAC + HBOT. In the APAP, NAC, and NAC + HBOT groups, liver injury was induced by oral administration of 1 g/kg APAP. The NAC group received 100 mg/kg NAC per day. NAC + HBOT group received intraperitoneal injection of 100 mg/kg/day NAC and were given HBOT at 2.8 ATA pressure with 100% O2 inhalation for 90 min every 12 h for 5 days. Rats in the sham group received distilled water only by gastric tube. All animals were killed on day 6 after APAP or distilled water administration. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, hepatic neopterin, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) levels were measured. There was a significant increase in serum AST and ALT activities in the APAP group compared with the sham group (in both p = 0.001). NAC and NAC + HBOT groups had significant decreases in hepatic neopterin, TNF-α, and IL-6 levels compared with the APAP group. APAP administration caused extensive hepatic necrosis. NAC and NAC + HBO treatments significantly reduced APAP-induced liver injury. Our results showed that the liver damage in APAP toxicity was attenuated by NAC and NAC + HBO treatments. NAC + HBOT exhibit hepatoprotective activity against APAP-induced liver injury in rats.

Aspartame induces angiogenesis in vitro and in vivo models

Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study is to evaluate the effect of aspartame on angiogenesis in vivo chick chorioallantoic membrane (CAM) and wound-healing models as well as in vitro 2,3-bis-2H-tetrazolium-5-carboxanilide (XTT) and tube formation assays. In CAM assay, aspartame increased angiogenesis in a concentration-dependent manner. Compared with the control group, aspartame has significantly increased vessel proliferation (p < 0.001). In addition, in vivo rat model of skin wound-healing study showed that aspartame group had better healing than control group, and this was statistically significant at p < 0.05. There was a slight proliferative effect of aspartame on human umbilical vein endothelial cells on XTT assay in vitro, but it was not statistically significant; and there was no antiangiogenic effect of aspartame on tube formation assay in vitro. These results provide evidence that aspartame induces angiogenesis in vitro and in vivo; so regular use may have undesirable effect on susceptible cases.

Evaluation of serum brain-derived neurotrophic factor levels in Type 2 diabetes mellitus patients with and without depressive symptoms

Dear Editor, We read with great interest the article byWang and He entitled ‘Levels of serum brain-derived neurotrophic factor in Type 2 diabetes mellitus patients with and without depressive symptoms’ in which the investigators reported that the serum brain-derived neurotrophic factor (BDNF) level was lower in Chinese Type 2 diabetes mellitus patients with or without depressive symptoms than that in healthy controls [1]. However, we think there are some points that should be discussed. BDNF, which is one of the neurotrophic factors, plays an important role on the synaptic and survival of neurons in the central nervous system. Several previous studies suggested that certain diseases such as hypertension, epilepsy, bipolar disease, major depression, schizophrenia, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, migraine, chronic periodontitis, rheumatoid arthritis, cardiovascular disease, atherosclerosis, metabolic syndrome, chronic kidney disease, colorectal cancer, lower respiratory tract infection, and atopic diseases (allergic rhinitis, atopic dermatitis) could affect serum BDNF levels [2,3]. In addition to the above diseases, clopidogrel, corticosteroids, antidepressants, statins, antitumor necrosis factor alpha, and aspirin could also alter BDNF levels [4,5]. Furthermore, dietary food supplements such as vitamin B12, vitamin A, vitamin E, folic acid, omega-3 fatty acids, zinc, and ginkgo biloba extracts could influence BDNF levels [6,7]. In this regard, without defining these contributing factors, interpreting the results is problematic. Serum BDNF levels were found to fluctuate during menstrual cycle. In follicular phase, BDNF levels were shown to be lower than that of luteal phase [8]. Begliuomini et al. [9] suggested that BDNF levels were higher in fertile women than that of women with amenorrhea or menopause. Maffioletti et al. [10] suggested that serum preparation procedure is a quite important issue for presenting robust methodology. Because of being stored in thrombocytes, BDNF level is highly affected by the duration of clotting process. They observed a progressive increase of serum BDNF levels, which reached 38% of the plateau value after 10 min, 91.8% after 30 min, and 100% after 1 h. Therefore, they suggested the minimum clotting duration for a correct serum BDNF dosage as 1 h. In the present study, the authors did not define sampling time, clotting period, or women’s menstrual status, which could cause falsely higher or lower serum BDNF levels. Therefore, standardization of methodology is essential for the measurement of serum BDNF. In conclusion, though this study contributes valuable information to the medical literature, clarifying these concerns will certainly provide a clearer picture when interpreting BDNF levels among participants.

Increased neopterin level and chitotriosidase activity in pregnant women with threatened preterm labor

Abstract Objectives: To determine whether the cellular inflammatory markers of activated macrophages, neopterin (NEO), chitotriosidase activity and the acute-phase inflammatory marker C-reactive protein (CRP) are elevated in pregnancy with threatened preterm labor (TPL). Methods: Thirty-two pregnant women with TPL and 32 women with uncomplicated pregnancy (UP) were included this study. The primary aim was to compare the NEO, chitotriosidase activity and CRP levels between women with TPL and women with UP. Results: NEO levels were all significantly elevated in patients with TPL compared to UP (median 25–75%; 9.61 [8.47–12.29] versus 4.46 [3.59–6.92], respectively; p < 0.001). Chitotriosidase activity was significantly elevated in pregnant women with TPL compared to UP (median 25–75%; 59.00 [38.00–87.25] versus 43.50 [23.25–65.25], respectively; p = 0.036). However, CRP levels were not different in women with TPL compared to UP (p = 0.573). Furthermore, a significant moderate negative correlation was found between delivery week and NEO level (r = −0.557, p = 0.001). However, a significant correlation was not seen between delivery week and chitotriosidase activity (r = −0.042, p = 0.741). Conclusions: Inflammatory markers such as NEO and chitotriosidase activity, which are markers of macrophages, are more elevated in pregnant women with TPL than in women with UP. These data suggest that there are striking increases in inflammation and cellular immune activation in TPL.

NFU1 gene mutation and mitochondrial disorders.

Mitochondrial respiratory chains consist of approximately 100 structural proteins. Thirteen of these structural proteins are encoded by mitochondrial DNA (mtDNA), and the others by nuclear DNA (nDNA). Mutation in any of the mitochondrial structural-protein related genes, regardless of whether they are in the nDNA or mtDNA, might cause mitochondrial disorders. In the recent past, new nuclear genes required for assembly, maintenance, and translation of respiratory chain proteins have been found. Mutation in these genes might also cause mitochondrial disorders (MD). NFU1 gene is one of such genes and has a role in the assembly of iron-sulfur cluster (ISC). ISCs are included in a variety of metalloproteins, such as the ferredoxins, as well as in enzymatic reactions and have been first identified in the oxidation-reduction reactions of mitochondrial electron transport. It is important to be aware of NFU1 gene mutations that may cause severe mitochondrial respiratory chain defects, mitochondrial encephalomyopathies and death, early in life.