Assessment of salivary miRNA, clinical, and demographic characterization in colorectal cancer diagnosis.

AIM: Colorectal cancer (CRC), as the third most frequent malignancy in the world, is the fourth major cause of cancer-related mortality. Its early detection contributes significantly to a reduction in mortality. The objective of this case-control research was to analyze the salivary expression of microRNA-29a (miR-29a) and microRNA-92a (miR-92a), and also to consider demographic, clinical, and nutritional habits for differentiation between CRC patients and healthy controls, especially in the early stages. METHOD: A standard checklist was used to obtain the demographic information, clinical features, and dietary habits of the case and control groups. Samplings of whole unstimulated saliva samples were obtained from 33 healthy persons and 42 CRC patients. Through real-time PCR, statistical analyses, and machine learning analyses, miR-29a and miR-92a salivary expression levels were evaluated. RESULTS: The mean salivary expression of miR-92a and miR-29a in CRC patients was significantly higher than in healthy controls (p < 0.001). The area under the receiver operating characteristic curve for miR-92a and miR-29a salivary biomarkers was 0.947 and 0.978, respectively. The sensitivity and specificity values for miR-92a were 95.24 % and 84.85 %, respectively, whereas sensitivity and specificity for miR-29a were equal to 95.20 % and 87.88 %, respectively. Multiple logistic regressions considering demographics, clinical features, and nutritional habits led to values of 95.35 % and 96.88 % as sensitivity and specificity, respectively, and machine learning analysis led to values of 88.89 % and 86.67 % as sensitivity and specificity, respectively. CONCLUSION: CRC could be accurately diagnosed based on miR-92a and miR-29a levels in saliva. Statistical analysis and machine learning might develop cost-effective models for the distinction of CRC using a noninvasive technique.

Different expression of DACT1, DACT2, and CYCLIN D1 genes in human colorectal cancer tissues and its association with clinicopathological characteristics.

Aberrant activation of Wnt pathway is linked to dysregulation of several genes. DACT1 and DACT2 are members of the DACT family that participate in antagonizing of the Wnt signaling cascade. Thus in this study, we assessed the mRNA levels of DACT1, DACT2, and CYCLIN D1 in 70 pairs of CRC tissues compared to the adjacent tissues. Determination of the mRNA levels of DACT1, DACT2, and CYCLIN D1 was done by Quantitative Real-Time PCR (qRT-PCR). The correlation between DACT1, DACT2, and CYCLIN D1 genes was also examined. Receiver operating characteristic (ROC) curves was plotted to assess the diagnostic power. The association between histopathological parameters and the DACT1, DACT2, and CYCLIN D1 genes was investigated. The expression levels of DACT1 and CYCLIN D1 were remarkably higher in CRC tissues compared to the adjacent tissues (p < 0.0001). However, the expression of DACT2 was decreased (p < 0.001). Our results showed a significant correlation between the expression of DACT1 and CYCLIN D1 (p < 0.0001). DACT1 (AUC = 0.74, p < 0.0001), DACT2 (AUC = 0.69, p < 0.0003), and CYCLIN D1 (AUC = 0.75, p < 0.0001) had good effectiveness in separation between CRC samples and adjacent tissues. We found a significant association between DACT1 expression with tumor site (p < 0.01). Also, a significant association was detected between DACT2 and CYCLIN D1 with tumor stage (p < 0.005 and p < 0.038, respectively). The findings suggested that DACT1 could function as an oncogene, whereas DACT2 was downregulated and can be considered as a tumor suppressor in CRC.

Low expression of LncRNA-CAF attributed to the high expression of HIF1A in esophageal squamous cell carcinoma and gastric cancer patients.

PURPOSE: Cancer-associated fibroblasts (CAFs) are major components of tumor microenvironment that stimulate ESCC and GC progression. The LncRNA-CAF, FLJ22447, is located in the vicinity of HIF1A, while their association remains unclear. This study aims to assess the FLJ22447 expression in the ESCC and GC patients and evaluate its association with the HIF1A gene. METHODS: Fresh ESCC and GC tumor samples and their adjacent non-tumor tissues were collected from patients who underwent surgery in Imam Khomeini Hospital, Tehran, Iran. The expression of FLJ22447, HIF1A, and VEGF was evaluated using qRT-PCR test. The association of their expression with tumor clinicopathological features in ESCC patients was assessed. System biology tools were then applied for the possible biological subsequences of the FLJ22447. RESULTS: A significant reduction in FLJ22447 expression was observed in ESCC and GC tissues than adjacent non-tumor tissues, while, the expression of HIF1A and VEGF were increased. Low expression of FLJ22447 was significantly correlated with HIF1A (P = 2.4e-73, R = 0.63) and VEGF (P = 0.00019, R = 0.15) expression. A significant relationship was detected between the high expression of HIF1A and tumor stages (I-II) and it was related to the reduced survival of ESCC patients. Conversely, increased VEGF expression was linked to the advanced stages (III-IV) and metastasis in ESCC. The analysis of FLJ22447-interacted proteins showed that MYC, JUN, SMRCA4, PPARG, AR, FOS, and CEBPA are the hub genes. These proteins were implicated in the cancer related pathways. Among them, SPI1, E2F1, TCF7L2, and STAT1 were significantly expressed in esophageal and gastric cancers that were functionally involved in the proliferation, apoptosis, and angiogenesis pathways in cancer. CONCLUSION: The results suggested that FLJ22447 may have a regulatory function on the HIF1A expression. We identified the FLJ22447-interacted proteins and their molecular function in cancer pathogenesis. Further research emphasis is to realize the association of FLJ22447 with its protein partners in progression of cancer. These may provide an insight into the FLJ22447 activity that could introduce it as a potential value in tumor gene therapy.

Evaluation of squamous cell carcinoma antigen 1 expression in oral squamous cell carcinoma (tumor cells and peritumoral T-lymphocytes) and verrucous carcinoma and comparison with normal oral mucosa.

BACKGROUND: Squamous cell carcinoma antigen (SCCA) is used as a prognostic marker for recurrence of squamous cell carcinoma in various sites, including head and neck. Studies suggest that its high serum levels are correlated to some clinical features, such as nodal metastasis. However, it is still unknown if high SCCA in patients with SCCA tissue expression in tumor cells are related to peripheral T-lymphocytes. Therefore, we did this study to evaluate SCCA expression in squamous cell carcinoma and verrucous carcinoma and to compare it with normal oral mucosa, also investigating the correlation between serum-based and tissue-based antigen levels. METHODOLOGY: In this study, the immunohistochemistry (IHC) technique was used to determine the SCCA1 expression pattern in 81 specimens divided into 3 groups, including oral squamous cell carcinoma, verrucous carcinoma, and normal oral mucosa. Serum-based and tissue-based antigen levels of 20 oral squamous cell carcinoma cases were compared by the western blot assay. SCCA expression was also evaluated and compared in both tumor cells and peripheral T-lymphocytes by the immunofluorescence assay. RESULTS: Our results showed that the SCCA levels in SCC specimens were significantly lower than in verrucous carcinoma and normal and hyperplastic oral mucosa specimens. We found no correlation between the IHC expression of SCCA and serum levels. SCCA was well expressed in both tumor cells and peripheral T-lymphocytes. CONCLUSION: Decreasing SCCA in SCC specimens suggested that SCC tumor cells may affect more than the serum levels of SCCA in some patients. In addition, expression of SCCA in peripheral T-lymphocytes showed that both tumor cells and T-lymphocytes may cause serum SCCA.

Long noncoding RNA LINC00978 acts as a potential diagnostic biomarker in patients with colorectal cancer.

Colorectal cancer (CRC) as a lethal malignancy has been associated with dysregulation of several genes and pathways. Long noncoding RNAs (lncRNAs) play an important role in gene expression regulation. In the current research, we aim to evaluate the expression of LINC00978 in CRC samples and adjacent tissues. Using Quantitative Real-Time PCR (qRT-PCR) method, we assessed the expression levels of LINC00978 and ß-catenin in 70 pairs of CRC and adjacent tissues. Moreover, the association between clinicopathological features and the LINC00978 expression levels was investigated. To assess the diagnostic power of LINC00978 expression in CRC, receiver operating characteristic (ROC) curve was plotted. The relationship between LINC00978 and ß-catenin expression levels was evaluated using correlation analysis. A markedly increased level of LINC00978 and ß-catenin expression levels was observed in CRC samples compared with adjacent tissues (P < 0.0001). No significant association was detected between LINC00978 expression level and the patient's clinicopathological features. The results of Pearson's correlation coefficient highlighted a positive correlation between LINC00978 and ß-catenin expression (r2 = 0.4695, P < 0.0001). According to the area under curve (AUC) value, LINC00978 expression differentiates CRC samples from the adjacent tissues (AUC = 0.81, P < 0.0001). The present results suggest that LINC00978 may play a critical role in CRC progression via Wnt pathway. The potential role of LINC00978 as a diagnostic biomarker needs to be further investigated in future studies.

Evaluation of squamous cell carcinoma antigen 1 expression in oral squamous cell carcinoma (tumor cells and peritumoral T-lymphocytes) and verrucous carcinoma and comparison with normal oral mucosa

Abstract Background: Squamous cell carcinoma antigen (SCCA) is used as a prognostic marker for recurrence of squamous cell carcinoma in various sites, including head and neck. Studies suggest that its high serum levels are correlated to some clinical features, such as nodal metastasis. However, it is still unknown if high SCCA in patients with SCCA tissue expression in tumor cells are related to peripheral T-lymphocytes. Therefore, we did this study to evaluate SCCA expression in squamous cell carcinoma and verrucous carcinoma and to compare it with normal oral mucosa, also investigating the correlation between serum-based and tissue-based antigen levels. Methodology: In this study, the immunohistochemistry (IHC) technique was used to determine the SCCA1 expression pattern in 81 specimens divided into 3 groups, including oral squamous cell carcinoma, verrucous carcinoma, and normal oral mucosa. Serum-based and tissue-based antigen levels of 20 oral squamous cell carcinoma cases were compared by the western blot assay. SCCA expression was also evaluated and compared in both tumor cells and peripheral T-lymphocytes by the immunofluorescence assay. Results: Our results showed that the SCCA levels in SCC specimens were significantly lower than in verrucous carcinoma and normal and hyperplastic oral mucosa specimens. We found no correlation between the IHC expression of SCCA and serum levels. SCCA was well expressed in both tumor cells and peripheral T-lymphocytes. Conclusion: Decreasing SCCA in SCC specimens suggested that SCC tumor cells may affect more than the serum levels of SCCA in some patients. In addition, expression of SCCA in peripheral T-lymphocytes showed that both tumor cells and T-lymphocytes may cause serum SCCA.

Over-expression of low-density lipoprotein receptor-related Protein-1 is associated with poor prognosis and invasion in pancreatic ductal adenocarcinoma.

BACKGROUND: Low-density lipoprotein receptor-Related Protein-1 (LRP-1) has been reported to involve in tumor development. However, its role in pancreatic cancer has not been elucidated. The present study was designed to evaluate the expression of LRP-1 in Pancreatic Ductal Adenocarcinoma Cancer (PDAC) as well as its association with prognosis. METHODS: Here, 478 pancreatic cancers were screened for suitable primary PDAC tumors. The samples were analyzed using qRT-PCR, western blotting, and Immunohistochemistry (IHC) staining as well as LRP-1 expression in association with clinicopathological features. RESULTS: The relative LRP-1 mRNA expression was up-regulated in 82.3% (42/51) of the PDAC tumors and its expression (3.72 ±â€¯1.25) was significantly higher than that in pancreatic normal margins (1.0 ±â€¯0.23, P < 0.05). This up-regulation was stage dependent (P < 0.05). A similar pattern of LRP-1 protein expression was discovered (P < 0.05). The high expression of LRP-1 in the PDAC tissues was strongly correlated with the low survival time (P = 0.001), TNM classification (P = 0.001), low differentiations status (P = 0.001), lymphatic invasion (P = 0.01) and Perineural Invasion (PNI) status (P = 0.001). CONCLUSIONS: Our finding for the first time revealed that LRP-1 expression inversely associated with poor prognosis and PNI in PDAC tumor.

Intraoperative consultation of central nervous system lesions. Frozen section, cytology or both?

OBJECTIVE: Frozen section is the traditional method of assessing central nervous system (CNS) lesions intraoperatively. Our aim is to determine the diagnostic accuracy of frozen section and/or cytological evaluation of CNS lesions in our center. STUDY DESIGN: A total of 157 patients with CNS lesions underwent open surgical biopsy or excision in our center during a period of 2 years (2012-2013). All specimens were studied cytologically; of these specimens, 146 cases were also examined by frozen section. Cytology and frozen section slides were studied separately by two general pathologists who were blind to final diagnoses. The final diagnoses were based on permanent sections and IHC studies. RESULTS: The accuracy rates of frozen section analysis and cytological evaluation were 87% and 86%, respectively. If the two methods were considered together, the accuracy rate improved to about 95%. CONCLUSIONS: Cytological evaluation is an acceptable alternative to frozen section analysis and also a great supplement to the diagnosis of CNS lesions.

Circulating levels of the miRNAs, miR-194, and miR-29b, as clinically useful biomarkers for colorectal cancer.

The microRNAs (miRNAs), miR-194 and miR-29b, have been shown to downregulate in colorectal cancer (CRC) and may identify and classify CRC patients as compared with those in control subjects. In the current study, we aimed to explore whether the serum levels of the miRNAs could be potential biomarkers for diagnosis and prognosis of CRC. A quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay was utilized to determine and compare serum levels of miR-194 and miR-29b in 55 patients with CRC and 55 control subjects. The correlations between levels of the miRNAs and clinicopathological stages of cancer were analyzed in patients. Receiver operating characteristic (ROC) curve and survival analyses were carried out, respectively, to determine diagnostic and prognostic values of the miRNAs. Serum levels of miR-194 and miR-29b were found to be significantly lower in CRC patients than those in control subjects (P < 0.0001). Moreover, serum levels of the miRNAs in patients were inversely correlated with the advanced TNM stages (P = 0.01). ROC curve and survival analyses revealed that reduced levels of the miRNAs could serve as diagnostic and prognostic biomarkers for patients with CRC (P = 0.0001). Serum levels of miR-194 and miR-29b may serve as potential biomarkers for diagnosis and prognosis of CRC.

Relationship of Amplification and Expression of the C-MYC Gene with Survival among Gastric Cancer Patients.

BACKGROUND: During the past decades, the incidence and mortality rate of stomach cancer has demonstrated a great decrease in the world, but it is still one of the most common and fatal cancers especially among men worldwide, including Iran. The MYC proto-oncogene, which is located at 8q24.1, regulates 15% of genes and is activated in 20% of all human tumors. MYC amplification and overexpression of its protein product has been reported in 15-30% of gastric neoplasias. The aim of this investigation was to find the relative efficacy of CISH (chromogenic in situ hybridization) or IHC (immunohistochemistry) in diagnosis and prognosis of gastric cancer, as well as the relationship of amplification and expression of C-MYC gene with patient survival. MATERIALS AND METHODS: In this cross-sectional study, 102 samples of gastric cancer were collected from patients who had undergone primary surgical resection at the Cancer Institute Hospital, Tehran University of Medical Sciences, from July 2009 to March 2014. All samples were randomly selected from those who were diagnosed with gastric adenocarcinomas. CISH and IHC methods were performed on all of them. RESULTS: Patients were classified into two groups. The first consisted of stage I and II cases, and the second of stage III and IV. Survival tests for both groups was carried out with referrnce to CISH test reults. Group II (stage III and IV) with CISH+ featured lower survival than those with CISH- (p=0.233), but group I (stage I and II) patients demonstrated no significant variation with CISH+ or CISH- (p=0.630). Kaplan-Meier for both groups was carried out with IHC test findings and showed similar results. This data revealed that both diffuse and intestinal types of gastric cancer occurred significantly more in men than women. Our data also showed that CISH+ patients (43%) were more frequent in comparison with IHC+ patients (14.7%). CONCLUSIONS: For planning treatment of gastric cancer patients, by focusing on expanding tumors, which is the greatest concern of the surgeons and patients, CISH is a better and more feasible test than IHC, in regard to sensitivity and specificity. Therefore, CISH can be used as a feasible test for tumor growth and prognosis in stage III and IV lesions. This study also indicated that C-MYC amplification in gastric cancer is correlated with survival in advanced stages.

Clinicopathologic features predicting involvement of non- sentinel axillary lymph nodes in Iranian women with breast cancer.

BACKGROUND: Almost half of the breast cancer patients with positive sentinel lymph nodes have no additional disease in the remaining axillary lymph nodes. This group of patients do not benefit from complete axillary lymph node dissection. This study was designed to assess the clinicopathologic factors that predict non-sentinel lymph node metastasis in Iranian breast cancer patients with positive sentinel lymph nodes. MATERIALS AND METHODS: The records of patients who underwent sentinel lymph node biopsy, between 2003 and 2012, were reviewed. Patients with at least one positive sentinel lymph node who underwent completion axillary lymph node dissection were enrolled in the present study. Demographic and clinicopathologic characteristics including age, primary tumor size, histological and nuclear grade, lymphovascular invasion, perineural invasion, extracapsular invasion, and number of harvested lymph nodes, were evaluated. RESULTS: The data of 167 patients were analyzed. A total of 92 (55.1%) had non-sentinel lymph node metastasis. Univariate analysis of data revealed that age, primary tumor size, histological grade, lymphovascular invasion, perineural invasion, extracapsular invasion, and the number of positive sentinel lymph nodes to the total number of harvested sentinel lymph nodes ratio, were associated with non-sentinel lymph node metastasis. After logistic regression analysis, age (OR=0.13; 95% CI, 0.02-0.8), primary tumor size (OR=7.7; 95% CI, 1.4-42.2), lymphovascular invasion (OR=19.4; 95% CI, 1.4- 268.6), extracapsular invasion (OR=13.3; 95% CI, 2.3-76), and the number of positive sentinel lymph nodes to the total number of harvested sentinel lymph nodes ratio (OR=20.2; 95% CI, 3.4-121.9), were significantly associated with non-sentinel lymph node metastasis. CONCLUSIONS: According to this study, age, primary tumor size, lymphovascular invasion, extracapsular invasion, and the ratio of positive sentinel lymph nodes to the total number of harvested sentinel lymph nodes, were found to be independent predictors of non-sentinel lymph node metastasis.

Association of plasma leptin, homocysteine and nitric oxide levels with the presence and unstability of coronary artery disease.

AIM: Leptin and total homocysteine (tHcy) may participate in the pathogenesis of coronary artery disease (CAD) through nitric oxide (NO) depletion. We sought to investigate whether leptin, tHcy and NO are suitable predictors of CAD. PATIENTS & METHODS: This study contained 50 control subjects and 50 stable and 50 unstable angina patients. Plasma leptin, tHcy and NO levels were determined using enzyme immunoassay, HPLC fluorescence and spectrophotometric methods, respectively. Other conventional risk factors were also determined. RESULTS: Leptin and tHcy levels were highest in unstable angina patients, followed by stable angina patients and then controls (p < 0.001). Controls had significantly higher NO than patients (p <0.001). Leptin and tHcy had a positive and NO a negative association with the presence of CAD. CONCLUSIONS: Some athrogenic effects of leptin may be mediated by affecting tHcy and NO levels. Plasma leptin, tHcy and NO levels showed significant contribution to CAD prediction and discrimination.

Serum paraoxonase 1 activity is associated with fatty acid composition of high density lipoprotein.

INTRODUCTION: Cardioprotective effect of high density lipoprotein (HDL) is, in part, dependent on its related enzyme, paraoxonase 1 (PON1). Fatty acid composition of HDL could affect its size and structure. On the other hand, PON1 activity is directly related to the structure of HDL. This study was designed to investigate the association between serum PON1 activity and fatty acid composition of HDL in healthy men. METHODS: One hundred and forty healthy men participated in this research. HDL was separated by sequential ultracentrifugation, and its fatty acid composition was analyzed by gas chromatography. PON1 activity was measured spectrophotometrically using paraxon as substrate. RESULTS: Serum PON1 activity was directly correlated with the amount of stearic acid and dihomo-gamma-linolenic acid (DGLA). PON1/HDL-C was directly correlated with the amount of miristic acid, stearic acid, and DGLA and was inversely correlated with total amount of ω 6 fatty acids of HDL. CONCLUSION: The fatty acid composition of HDL could affect the activity of its associated enzyme, PON1. As dietary fats are the major determinants of serum lipids and lipoprotein composition, consuming some special dietary fatty acids may improve the activity of PON1 and thereby have beneficial effects on health.

Evaluation of the effects of weak and moderate static magnetic fields on the characteristics of human low density lipoprotein in vitro.

It has been suggested that exposure to electromagnetic fields may be a risk factor for cardiovascular disease in humans. Low density lipoprotein (LDL) modifications such as peroxidation and aggregation have been implicated in the pathogenesis of atherosclerosis. The present study investigated the effects of weak (0.125-0.5 mT) and moderate (1-4 mT) static magnetic fields (SMFs) on LDL oxidation, aggregation and zeta potential in vitro. Our results demonstrated that magnetic flux densities of 0.25 and 0.5 mT decreased, and magnetic flux densities of 3 and 4 mT increased the zeta potential and LDL oxidation in comparison with the control samples. All doses of SMFs increased the LDL aggregation in a time- and dose-dependent manner. It is concluded that SMFs can alter the susceptibility of LDL to oxidation and this alteration is dependent on the applied magnetic flux density. The SMF, in addition to its role in the production and stabilization of free radicals and promotion of lipid peroxidation, may influence the metabolism of lipoproteins and their interaction with other molecules such as apolipoproteins, enzymes and receptors through the alteration of the LDL zeta potential and its particles tendency to aggregation.

Associations between high density lipoprotein mean particle size and serum paraoxonase-1 activity.

BACKGROUND: High density lipoprotein (HDL) particles are heterogeneous in composition, structure, size, and may differ in conferring protection against cardiovascular disease. HDL associated enzyme, paraoxonase-1 (PON1), has an important role in attenuation of atherogenic low density lipoprotein (LDL) oxidation. The aim of this study was to investigate the associations between HDL particle size and PON1 activity in relation to serum HDL cholesterol (HDL-C) levels. MATERIALS AND METHODS: One hundred and forty healthy subjects contributed to this study. HDL was separated by sequential ultracentrifugation and its size was estimated by dynamic light scattering. Paraoxonase activity was measured spectrophotometrically using paraoxon as substrate. RESULTS: Results of this study showed that PON1 activity had negative correlations with HDL mean particle size (r = -0.22, P < 01), HDL2/HDL3 ratio, and serum HDL-C levels (r = -0.25, P < 0.01). HDL mean particle size and HDL2/HDL3 ratio had negative correlation with body mass index (BMI), waist hip ratio (WHR), and serum triglyceride (TG) levels, and positive correlation with serum HDL-C levels. Serum HDL-C levels had significant positive correlations with age, total cholesterol (TC), and apolipoprotein A-I (apo A-I) and significant negative correlation with BMI, WHR, and TG. CONCLUSION: Based on the results of this study, determination of HDL mean particle size beside the serum PON1 activity may help to better understand the CAD risks, pathogenesis, and prognosis, and may also help to design therapeutic protocols toward beneficial modifications of HDL characteristics.

Comparison of different techniques for hemostasis in a rabbit model of hypospadias repair.

PURPOSE: We compared the cellular and ultrastructural changes in the urethral wall following application of different hemostasis techniques in a rabbit model of hypospadias surgery. MATERIALS AND METHODS: Rabbits were allocated into 5 groups. In group 1 animals underwent surgery without application of any hemostasis technique; in group 2 continuous penile tourniquet was applied for 30 minutes; in group 3, 3 intermittent periods of 10-minute penile tourniquet were applied with 3-minute intervals of reperfusion; in group 4 epinephrine was injected to maintain a 30-minute period of hemostasis; and in group 5 epinephrine vehicle (normal saline) was injected during the procedure. Early urothelium ultrastructural damage was studied 1 hour postoperatively with electron microscopy. Apoptotic damage and histopathological changes were determined 48 hours following the procedure. Late onset complications were assessed with retrograde urethrography and evaluation of tissue fibrosis at 8 weeks postoperatively. RESULTS: Electron microscope studies demonstrated urothelium ultrastructural damage in all hemostasis groups compared to controls. However, the changes were most prominent in group 4. The apoptosis index of urethral wall myocytes in groups 1 and 5 was significantly lower compared to other groups. Moreover, the number of apoptotic myocytes in epinephrine injected animals was significantly higher than in the continuous or intermittent tourniquet group as well as the normal saline injected group. At 8 weeks postoperatively collagen deposition in the urethral wall of rabbits in group 4 was higher than that in group 1. Although urethrocutaneous fistula was found in only 1 rabbit in group 4, the difference was not significant. CONCLUSIONS: Hemostasis techniques applied for maintaining a bloodless surgical field during hypospadias repair may lead to ischemia/reperfusion tissue damage in the urethral wall. Our findings suggest that epinephrine injection may result in more prominent cellular changes compared to tourniquet techniques. However, further experimental and human studies are required to draw a firm conclusion.