Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.

Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.

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Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2

Urinary bladder carcinoma contributes to 4% of newly diagnosed oncological diseases in the Czech Republic. Biomarkers for its early non-invasive detection are therefore highly desirable. Urine seems to be an ideal source of such biomarkers due to the content of cell-free nucleic acids, especially microRNAs (miRNAs).To find potential biomarkers among miRNAs in urine supernatant, we examined in total 109 individuals (36 controls and 73 bladder cancer patients) in three phases. In the first - discovery - phase, microarray cards with 381 miRNAs were used for miRNA analysis of 13 controls and 46 bladder cancer patients. In the second - verification - phase, the results of this first phase were verified on the same groups of subjects by single-target qPCR assays for the selected miRNAs. For the third - validation - phase, new independent samples of urine supernatant (23 controls and 27 bladder cancer patients) were analyzed using single-target qPCR assays for 13 verified in the previous phase. The results of all phases were normalized to miR-191, miR-28-3p, and miR-200b, which were selected as suitable for our study by the qBase+®.We found that miR-125b, miR-30b, miR-204, miR-99a, and miR-532-3p are significantly down-regulated in patients' urine supernatant. In our experiments, the analysis of miR-125 levels provided the highest AUC (0.801) with 95.65% specificity and 59.26% sensitivity, the analysis of miR-99a lead to AUC (0.738) with 82.61% specificity and 74.07% sensitivity. We demonstrate that levels of these miRNAs could potentially serve as promising diagnostic markers for the non-invasive diagnostics of bladder cancer.

Introduction: Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. Material and Methods: In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. Results: The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. Conclusion: Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer.

SHB is an

The aim of the study was to define specific genetic profile in Ta and T1 urinary bladder carcinoma patients with and without recurrence by gene expression microarrays. Eleven patients with the time to recurrence shorter than one year (patients with recurrence) and 11 patients with time to recurrence longer than 4 years (patients without recurrence) were enrolled.Data from microarrays were subjected to a panel of statistical analyses to identify bladder cancer recurrence-associated gene signatures. Initial screening using the GeneSpring and Bioconductor software tools revealed a putative set 47 genes differing in gene expression in both groups. After the validation, 33 genes manifested significant differences between both groups. The significant expression was observed in the group of patients without recurrence by 30 genes of which the highest differences were detected by ANXA1, ARHGEF4, FLJ32252, GNE, NINJ1, PRICKLE1, PSAT1, RNASE1, SPTAN1, SYNGR1, TNFSF15, TSPAN1, and WDR34. These genes code for signal transduction, vascular remodeling and vascular endothelial growth inhibition mainly. In the group with recurrence, 3 genes had significant differences, the highest differences were identified by two genes (PLOD2 and WDR72).Loci of genes with significant changes of gene expression were located on characteristic chromosomes for bladder cancer: 7 loci on chromosome 9, 8 loci on chromosomes 1, 2, 3, 12, 14, 15, 16, and 22. We have selected and validated 15 genes that are differentially expressed in superficial bladder cancer. We hope that this cohort of genes will serve as a promising pool of candidate biomarkers for early stage bladder cancer. Our results indicate that it may be possible to identify patients with a low and high risk of disease recurrence at an early stage using a molecular profile. Key words: bladder cancer, non-muscle invasive urothelial tumors, gene expression microarraysBladder cancer (BC) is the sixth most frequent solid tumor in men and thirteenth in women in Czech Republic with 1827 and 650 new cases in 2005, respectively [1] and 375.000 new cases and 145.000 deaths worldwide annually [2]. Urothelial carcinoma (UC) is a heterogenous neoplasm manifesting either non-muscle invasive bladder cancer (NMIBC) -Ta, T1 and Tis by approximatelly 75 % newly diagnosed cases or muscle invasive (T2-T4) and metastatic tumor (25 %) [3,4]. After initial treatment of NMIBC by transurethral resection (TURB) up to 80 % of patients develop recurrences. From 10 % to 15 % of them progress to muscle invasive cancer [5,6]. For treatment and follow up of patients it is crucial to predict the recurrence and progression potential of non-muscle invasive bladder cancer. Therefore new methods are engaged to identify new prognostic markers based on the molecular nature of the tumor development and recurrence [7,8].The objective of our study was to identify the genes differing in gene expression in tumors with and without recurrence. Among the genes it might be determined a gene or several genes serving as factors for furt...