Small non-coding RNA profiling in breast cancer: plasma U6 snRNA, miR-451a and miR-548b-5p as novel diagnostic and prognostic biomarkers.

BACKGROUND: Breast cancer is a leading cause of cancer-related death in women. Most cases are invasive ductal carcinomas of no special type (NST breast carcinomas). METHODS AND RESULTS: In this prospective, multicentric biomarker discovery study, we analyzed the expression of small non-coding RNAs (mainly microRNAs) in plasma by qPCR and evaluated their association with NST breast cancer. Large-scale expression profiling and subsequent validations have been performed in patient and control groups and compared with clinicopathological data. Small nuclear U6 snRNA, miR-548b-5p and miR-451a have been identified as candidate biomarkers. U6 snRNA was remarkably overexpressed in all the validations, miR-548b-5p levels were generally elevated and miR-451a expression was mostly downregulated in breast cancer groups. Combined U6 snRNA/miR-548b-5p signature demonstrated the best diagnostic performance based on the ROC curve analysis with AUC of 0.813, sensitivity 73.1% and specificity 82.6%. There was a trend towards increased expression of both miR-548b-5p and U6 snRNA in more advanced stages. Further, increased miR-548b-5p levels have been partially associated with higher grades, multifocality, Ki-67 positivity, and luminal B rather than luminal A samples. On the other hand, an association has been observed between high miR-451a expression and progesterone receptor positivity, lower grade, unifocal samples, Ki-67-negativity, luminal A rather than luminal B samples as well as improved progression-free survival and overall survival. CONCLUSIONS: Our results indicated that U6 snRNA and miR-548b-5p may have pro-oncogenic functions, while miR-451a may act as tumor suppressor in breast cancer.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

Genome-wide miRNA profiling in plasma of pregnant women with down syndrome fetuses.

Down syndrome (DS) is one of the most common causes of intellectual disability and new approaches allowing its rapid and effective prenatal detection are being explored. In this study, we investigated the diagnostic potential of plasma microRNAs (miRNAs). This study builds upon our previous study in DS placentas, where seven miRNAs were found to be significantly up-regulated. A total of 70 first-trimester plasma samples from pregnant women were included in the present study (35 samples with DS fetuses; 35 with euploid fetuses). Genome-wide miRNA profiling was performed in the pilot study using Affymetrix GeneChip™ miRNA 4.1 Array Strips (18 samples). Selected miRNAs were then analysed in the validation study using quantitative reverse transcription PCR (RT-qPCR; 52 samples). Based on the current pilot study results (12 miRNAs), our previous research on chorionic villi samples (7 miRNAs) and the literature (4 miRNAs), a group of 23 miRNAs was selected for the validation study. Although the results of the pilot study were promising, the validation study using the more sensitive RT-qPCR technique and a larger group of samples revealed no significant differences in miRNA profiles between the compared groups. Our results suggest that testing of the first-trimester plasma miRNAs is probably not suitable for non-invasive prenatal testing (NIPT). Different results could be theoretically achieved at later gestational ages; however, such a result probably would have limited use in clinical practice.

Ovarian Cancer: Differentially Expressed microRNAs in Tumor Tissue and Cell-Free Ascitic Fluid as Potential Novel Biomarkers.

Ovarian cancer is the deadliest gynecologic cancer. The large-scale microRNA (miRNA) expression profiling and individual miRNA validation was performed to find potential novel biomarkers for ovarian cancer. The most consistent overexpression of miRs-200b-3p, 135 b-5p and 182-5p was found in both ascitic fluid and tumors and suggests their potential as oncogenes. miR-451a was consistently underexpressed so may be a tumor suppressor. Results were inconsistent for miR-204-5p, which was overexpressed in ascitic fluid but underexpressed in tumor tissue. miR-203a-3p was generally overexpressed but this failed to be proved in independent sample set in tissue validation.

The utility of massively parallel sequencing for posterior polymorphous corneal dystrophy type 3 molecular diagnosis.

The aim of this study was to identify the molecular genetic cause of disease in posterior polymorphous corneal dystrophy (PPCD) probands of diverse origin and to assess the utility of massively parallel sequencing in the detection of ZEB1 mutations. We investigated a total of 12 families (five British, four Czech, one Slovak and two Swiss). Ten novel and two recurrent disease-causing mutations in ZEB1, were identified in probands by Sanger (n = 5), exome (n = 4) and genome (n = 3) sequencing. Sanger sequencing was used to confirm the mutations detected by massively parallel sequencing, and to perform segregation analysis. Genome sequencing revealed that one proband harboured a novel ∼0.34 Mb heterozygous de novo deletion spanning exons 1-7 and part of exon 8. Transcript analysis confirmed that the ZEB1 transcript is detectable in blood-derived RNA samples and that the disease-associated variant c.482-2A>G leads to aberrant pre-mRNA splicing. De novo mutations, which are a feature of PPCD3, were found in the current study with an incidence rate of at least 16.6%. In general, massively parallel sequencing is a time-efficient way to detect PPCD3-associated mutations and, importantly, genome sequencing enables the identification of full or partial heterozygous ZEB1 deletions that can evade detection by both Sanger and exome sequencing. These findings contribute to our understanding of PPCD3, for which currently, 49 pathogenic variants have been identified, all of which are predicted to be null alleles.

Schnyder corneal dystrophy and associated phenotypes caused by novel and recurrent mutations in the UBIAD1 gene.

BACKGROUND: The purpose of this study was to identify the genetic cause and describe the clinical phenotype of Schnyder corneal dystrophy (SCD) in six unrelated probands. METHODS: We identified two white Czech, two white British and two South Asian families with a clinical diagnosis of SCD. Ophthalmic assessment included spectral domain optical coherence tomography (SD-OCT) of one individual with advanced disease, and SD-OCT and confocal microscopy of a child with early stages of disease. UBIAD1 coding exons were amplified and Sanger sequenced in each proband. A fasting serum lipid profile was measured in three probands. Paternity testing was performed in one family. RESULTS: A novel heterozygous c.527G>A; p.(Gly176Glu) mutation in UBIAD1 was identified in one Czech proband. In the second Czech proband, aged 6 years when first examined, a previously described de novo heterozygous c.289G>A; p.(Ala97Thr) mutation was found. Two probands of South Asian descent carried a known c.305G>A; p.(Asn102Ser) mutation in the heterozygous state. Previously reported heterozygous c.361C>T; p.(Leu121Phe) and c.308C>T; p.(Thr103Ile) mutations were found in two white British families. Although crystalline deposits were present in all probands the affected area was small in some individuals. Corneal arcus and stromal haze were the most prominent phenotypical feature in two probands. In the Czech probands, SD-OCT confirmed accumulation of reflective material in the anterior stroma. Crystalline deposits were visualized by confocal microscopy. Mild dyslipidemia was found in all three individuals tested. CONCLUSION: Although de novo occurrence of mutations in UBIAD1 is extremely rare, SCD should be considered in the differential diagnosis of bilateral corneal haze and/or crystal deposition, especially in children.

Serum microRNA-196 and microRNA-200 in pancreatic ductal adenocarcinoma of patients with diabetes mellitus.

BACKGROUND/OBJECTIVES: Our aim was to compare expressions of 6 microRNAs (miRNAs) in patients with pancreatic ductal adenocarcinoma (PAC) and non-cancer patients, moreover according to the presence or absence of diabetes mellitus. METHODS: Expressions of miRNA-192, -196, -200, -21, -30 and -423 were measured in 77 patients with PAC and 64 non-cancer patients (34 patients with type 2 DM and 30 control persons). 60 patients with PAC (78%) had DM or prediabetes and it was of new-onset (less than 2 years before the cancer diagnosis) in 44 out of them. RESULTS: The expressions of all microRNAs were 1.4-3.7 times higher (significantly) in the PAC group compared to non-cancer patients. No difference was found between PAC diabetic and PAC non-diabetic patients. MicroRNA-200 was significantly higher in PAC patients with significant body weight loss against those without weight loss. Adding miRNA-196 and -200 to the current marker CA 19-9 improved the discriminative ability of the test (compared to CA 19-9 alone). CONCLUSION: MicroRNA-196 and -200 could be used as additional markers in PAC diagnosis.

Differentially expressed miRNAs in trisomy 21 placentas.

OBJECTIVE: Molecular pathogenesis of Down syndrome (DS) is still incompletely understood. Epigenetic mechanisms, including miRNAs gene expression regulation, belong to potential influencing factors. The aims of this study were to compare miRNAs expressions in placentas with normal and trisomic karyotype and to associate differentially expressed miRNAs with concrete biological pathways. METHODS: A total of 80 CVS samples - 41 with trisomy 21 and 39 with normal karyotype - were included in our study. Results obtained in the pilot study using real-time PCR technology and TaqMan Human miRNA Array Cards were subsequently validated on different samples using individual TaqMan miRNA Assays. RESULTS: Seven miRNAs were verified as upregulated in DS placentas (miR-99a, miR-542-5p, miR-10b, miR-125b, miR-615, let-7c and miR-654); three of these miRNAs are located on chromosome 21 (miR-99a, miR-125b and let-7c). Many essential biological processes, transcriptional regulation or apoptosis, were identified as being potentially influenced by altered miRNA levels. Moreover, miRNAs overexpressed in DS placenta apparently regulate genes involved in placenta development (GJA1, CDH11, EGF, ERVW-1, ERVFRD-1, LEP or INHA). CONCLUSION: These findings suggest the possible participation of miRNAs in Down syndrome impaired placentation and connected pregnancy pathologies. © 2016 John Wiley & Sons, Ltd.

Comparison of MicroRNA Content in Plasma and Urine Indicates the Existence of a Transrenal Passage of Selected MicroRNAs.

MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer. To examine the spectrum of miRNAs in the cell-free fraction of urine, TaqMan Human miRNA Array Card A v.2.1 was used. A set of 30 miRNAs were found that are constantly present in urine supernatants independently of sex, age and health status of the subjects. We compared this set with miRNAs found in plasma, expressed in kidney and genito-urinary tract. Our results indicate that some miRNA could be transferred from the circulation into urine.

Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with 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.

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.

[miRNA as a new marker of diabetes mellitus and pancreatic carcinoma progression]. / miRNA jako nový ukazatel u diabetes mellitus a u rozvoje karcinomu pankreatu.

Pancreatic cancer is a disease with increasing incidence and high (and nearly unchanged) lethality that is caused mainly due to its late diagnosis. Risk factors for neoplastic transformation are especially chronic pancreatitis, diabetes mellitus, but also obesity and smoking. The search for suitable early markers becomes a key element of research in this area. Such markers could be microRNAs, short single-stranded RNA molecules functioning as regulators of translation. This article serves as a review of contemporary evidence of microRNA in diabetes mellitus and pancreatic cancer.

[miRNA-192, miRNA-21 and miRNA-200: new pancreatic cancer markers in diabetic patients?]. / miRNA-192, miRNA-21 a miRNA-200: nové markery karcinomu pankreatu u diabetiku?

INTRODUCTION: Newly-onset diabetes mellitus (DM) in middle-aged and older people may be an early symptom of pancreatic cancer (PC). However, sensitive markers for PC are still missing. MicroRNAs (miRNAs) play an important role in a cell response regulation. Significant changes in miRNA expressions were observed in cancers. Our goal was to compare expressions of selected miRNAs in patients with PC, DM and controls. METHODS: We enrolled 74 patients with PC (42/32, with/without DM), 29 type 2 diabetic patients and 17 controls. MicroRNA was determined in serum of all examined subjects. In 9 patients with PC the tumor was resected subsequently and after 3 months the measurements were repeated. We analyzed the expressions of 8 miRNAs that we had identified in a previous pilot study (miR-21, miR-30, miR-191, miR-192, miR-196, miR-200, miR-423, miR-454). RESULTS: MicroRNA expressions were significantly higher in patients with PC than in DM and controls: miR-192: 1.6 (1.2 to 2.0) vs 0.3 (0.2-0.4) vs 0.3 (0.2 to 0.5), p < 0.00001; miR-21: 1.4 (1.2-1.7) vs 0.3 (0.2 to 0.5) vs 0.5 (from 0.4 to 0.7), p < 0.00001, miR-200: 1.6 (1.1 to 2.3) vs 0.3 (0.3-0.4) vs 0.3 (0.2 to 0.4), p < 0.00001. No difference was observed between DM and controls, as well as between diabetic and non-diabetic patients within the PC group. There were no significant differences in miRNA expressions in 9 patients after pancreatic surgery. But there were significant interindividual differences. CONCLUSION: Our data shows that miR-21, miR-192 and miR-200 could be used as new diagnostic markers for pancreatic cancer. A dynamics of these miRNAs could serve as a prognostic marker in patients after cancer removal. Futher prospective studies with newly-onset diabetic patients with no signs of malignancy will be needed to validate if suggested miRNAs could be used as early markers as well.

Investigator® Argus X-12 study on the population of Czech Republic: comparison of linked and unlinked X-STRs for kinship analysis.

DNA samples of 523 unrelated anonymized individuals (307 males and 216 females) born and living in the Czech Republic were genotyped using Investigator® Argus X-12 system in the following loci localized in four linkage groups: DXS10148, DXS10135, DXS8378, DXS7132, DXS10079, DXS10074, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134, DXS742. Haplotype frequencies were calculated for each LG (Linkage Group). The frequency of most common haplotype was 0.016, 0.036, 0.042, and 0.023 for LG1, LG2, LG3, and LG4, respectively. The combined power of discrimination was more than 0.999999999 both for female and male samples. The mean exclusion chance was 0.99999999 (trios) and 0.999999 (duos). Informativity and suitability of Investigator® Argus X-12 for kinship determination was assessed by computing in several female-female duos using LR (Likelihood Ratio) determination for autosomal STR (PowerPlex ESI-17), linked (Investigator® Argus X-12 system), and unlinked (X-STR Decaplex) X-STR kits. Investigator® Argus X-12 proved to be very useful for sibship determination, since its LR values were relatively similar to LR for autosomal STR kit. This work presents the first population data for Investigator® Argus X-12 system in the Czech Republic.

Application of the new insertion-deletion polymorphism kit for forensic identification and parentage testing on the Czech population.

Insertion-deletion polymorphisms (INDELs) are diallelic markers derived from a single mutation event. Their low mutation frequency makes them suitable for forensic and parentage testing. The examination of INDELs thus combines advantages of both short tandem repeats (STR) and single nucleotide polymorphisms (SNP). This type of polymorphisms may be examined using as small amplicon size as SNP (about 100 bp) but could be analyzed by techniques used for routine STR analysis. For our population study, we genotyped 55 unrelated Czech individuals. We also genotyped 11 trios to analyze DIPplex Kit (QIAGEN, Germany) suitability for parentage testing. DIPplex Kit contains 30 diallelic autosomal markers. INDELs in DIPplex Kit were tested with linkage disequilibrium test, which showed that they could be treated as independent markers. All 30 loci fulfill Hardy-Weinberg equilibrium. There were several significant differences between Czech and African populations, but no significant ones within European population. Probability of a match in the Czech population was 1 in 6.8 × 10(12); combined power of discrimination was 99.9999999999%. Average paternity index was 1.13-1.77 for each locus; combined paternity index reached about 27,000 for a set of 30 loci. We can conclude that DIPplex kit is useful as an additional panel of markers in paternity cases when mutations in STR polymorphisms are present. For application on degraded or inhibited samples, further optimization of buffer and primer concentrations is needed.

Methylation status of immune response genes promoters in cell-free DNA differs in hemodialyzed patients with diabetic nephropathy according to the intensity of anemia therapy.

BACKGROUND: Anemia is a major complication of end-stage renal disease. Hemodialysis itself is regarded as a stimulus activating inflammation. Pro-inflammatory cytokines are able to suppress erythropoiesis. In this pilot study, we analyzed the changes in methylation status of promoters of immune response genes in cell-free DNA to detect the differences between diabetic subjects (n = 18) with different therapeutic doses of recombinant erythropoietin. METHODS: The extent of promoter methylation of 24 genes in plasma cell-free DNA was examined before and after hemodialysis using EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity (Qiagen). RESULTS: The patients with higher methylation status of gene sequences IL13RA1, IL15, EDG3 and INHA in interdialytic interval were significantly overrepresented in the group with none or mild anemia therapy. CONCLUSION: The results are in agreement with the fact that IL13 and IL15 are known inhibitors of erythropoiesis and with considered immunomodulatory role of cell-free DNA.

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

Snail Track Lesion with Flat Keratometry in Anterior Segment Dysgenesis Caused by a Novel FOXC1 Variant.

We report the phenotype of a 15-year-old female patient with anterior segment dysgenesis (ASD) caused by a novel heterozygous loss-of-function FOXC1 variant. The proband underwent an ophthalmic examination as well as a molecular genetic investigation comprising exome sequencing, a single nucleotide polymorphism array to access copy number and Sanger sequencing to exclude non-coding causal variants. There was bilateral mild iris hypoplasia with pupil deformation and iridocorneal adhesions. In addition to these features of ASD, the corneas were flat, with mean keratometry readings of 38.8 diopters in the right eye and 39.5 diopters in the left eye. There was a snail track lesion of the left cornea at the level of the Descemet membrane. The central corneal endothelial cell density was reduced bilaterally at 1964 and 1373 cells/mm2 in the right and left eyes, respectively. Molecular genetic analysis revealed that the proband was a carrier of a novel heterozygous frameshifting variant in FOXC1, c.605del p.(Pro202Argfs*113). Neither parent had this change, suggesting a de novo origin which was supported by paternity testing. We found no possibly pathogenic variants in the other genes associated with posterior corneal dystrophies or ASD. Further studies are warranted to verify whether there is a true association between snail track lesions, corneal flattening, and pathogenic variants in FOXC1.

Resveratrol attenuates lipopolysaccharide-induced hepatitis in D-galactosamine sensitized rats: role of nitric oxide synthase 2 and heme oxygenase-1.

The goal of study was directed to investigate the effects of resveratrol (RES) pretreatment on the enhancing action of D-galactosamine (D-GalN; 800 mg/kg) on lipopolysaccharide (LPS; 0.5 microg/kg) inducing liver failure in rats. Liver function was assessed by determination of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alpha-glutathione S-transferase (alpha GST) and bilirubin (BILI). Plasma NO(2)(-) was assessed by NO(2)(-)/NO(3)(-) colorimetric kit. The estimation of nonenzymatic and enzymatic antioxidants (glutathione and catalase) was performed in plasma and liver homogenate. Lipid peroxidation was evaluated by the thiobarbituric acid reacting substances (TBARS) and the conjugated dienes (CD). Morphological examinations using light and electron microscopy were performed. Observations related to pharmacological increases of inducible nitric oxide synthase (NOS-2)/nitric oxide (NO) and inducible heme oxygenase (HO-1) in fulminant hepatic failure and modulation by resveratrol were followed up by real-time reverse transcription PCR (RT-PCR) in liver tissue. In the present study we found that among the mechanisms responsible for the hepatoprotective effect of resveratrol in the LPS/D-GalN liver toxicity model are reduction in NO, downregulation of NOS-2, modification of oxidative stress parameters and modulation of HO-1 which led to overall improvement in hepatotoxic markers and morphology after the hepatic insult.

Effects of resveratrol pretreatment on tert-butylhydroperoxide induced hepatocyte toxicity in immobilized perifused hepatocytes: involvement of inducible nitric oxide synthase and hemoxygenase-1.

The aim of this work was to study the effects of resveratrol (RES) as compared to silymarin (SM) pretreatments on tert-butylhydroperoxide (tBH) induced apoptotic/necrotic markers in hepatocytes. Hepatocyte in cultures (48 h) and in perifused immobilized agarose threads (5h) were used as cellular systems. Hepatocyte apoptosis was estimated morphologically using Annexin-V combined with propidium iodide, or toluidine blue staining. Hepatocyte viability and functionality were evaluated by ALT and urea synthesis. Nitric oxide (NO) and carbon monoxide involvements were also examined. Resveratrol and silymarin reduced tBH-induced hepatocyte toxic effects in short term experiments (5h) as measured by a significant reduction in ALT and NO increase produced by tBH. Both inducible nitric oxide synthase (NOS-2) and hemoxygenase-1 (HO-1) gene expression were increased by tBH and reduced by both RES and SM pretreatments. Morphologically, there were ameliorations in both apoptotic and necrotic markers under RES treatment and were similar to biochemical findings. In addition, RES improved hepatocyte stability in both cellular systems. It may be concluded that resveratrol and sylimarin ameliorative effects on tBH hepatocyte toxicity are comparable; involve NOS-2 and HO-1 expression and should be re-evaluated in various in vitro and in vivo experimental conditions.