Immunogenetic Prediction of VDR Gene SNPs: Lack of Association with Susceptibility to Type 1 Diabetes in Jordanian Patients.

PURPOSE: The interaction of Vitamin D and its receptor plays a crucial role in immune modulation. Therefore, the relationship between the pathogenesis of type 1 diabetes and the genetic variants of Vitamin D receptor, which is involved in the activity of Vitamin D, was studied extensively in different populations. The association of Vitamin D receptor gene polymorphisms with predisposition to type 1 diabetes revealed controversial and inconclusive results. The aim of this study was to examine the association of four Vitamin D receptor polymorphisms with type 1 diabetes in Jordanian patients. PATIENTS AND METHODS: Analysis of the single nucleotide polymorphisms FokI (rs2228570), ApaI (rs7975232), TaqI (rs731236) and BsmI (rs1544410) in 100 Jordanian volunteers (50 control and 50 Type 1 diabetes patients) was performed using the highly specific New Generation Sequencing technology. RESULTS: The distribution of allele, genotype as well as haplotype frequencies exhibited no significant (P > 0.05) differences between type 1 diabetes patients and controls. Furthermore, no differences (P > 0.05) in the frequency of the genotypes of the Vitamin D receptor genetic variants were found in relation to the age of disease onset. CONCLUSION: These findings suggest these four single nucleotide polymorphisms of the Vitamin D receptor gene seem not to be associated with type 1 diabetes predisposition in Jordanian patients. Further wide genome studies are recommended to detect other genetic variant associations with type 1 diabetes among Jordanians.

In vivo investigation on the chronic hepatotoxicity induced by sertraline.

Although sertraline is widely prescribed as relatively safe antidepressant drug, hepatic toxicity was reported in some patients with sertraline treatment. The present study was conducted to investigate the morphometric, hepatotoxicity, and change in gene expression of drug metabolizing enzymes. Male healthy adult rabbits (Oryctolagus cuniculus) ranging from 1050 to 1100 g were exposed to oral daily doses of sertraline (0, 1, 2, 4, 8 mg/kg) for 9 weeks. The animals were subjected to morphometric, hepatohistological, histochemical and quantitative real-time polymerase chain reaction analyses. Sertraline chronic exposure induced morphometric changes and provoked histological and histochemical alterations including: hepatocytes hydropic degeneration, necrosis, nuclear alteration, sinusoidal dilation, bile duct hyperplasia, inflammatory cells infiltration, portal vessel congestion, Kupffer cells hyperplasia, portal fibrosis and glycogen depletion. In addition, the gene expression of drug and arachidonic acid metabolizing enzymes were reduced significantly (p value <0.05). The most affected genes were cyp4a12, ephx2, cyp2d9 and cyp1a2, demonstrating 5 folds or more down-regulation. These findings suggest that chronic sertraline treatment induced toxic histological alterations in the hepatic tissues and reduced the gene expression of drug metabolizing enzymes. Patients on chronic sertraline treatment may be on risk of hepatotoxicity with reduced capacity to metabolize drugs and fatty acids.

Ameliorative effect of propolis against hepatorenal alterations induced by methotrexate: morphohistopathological study / Efecto protector del propóleo en las alteraciones hepatorrenales inducidas por el metotrexato: estudio morfohistopatológico

Methotrexate (MTX) is widely used in the treatment of some forms of cancer but having severe side effects. The present work aimed to investigate the protective role of propolis treatment against alterations induced by MTX on the hepatic and renal tissues. Rabbits were exposed to MTX (0.25 mg/kg), with or without propolis (50 mg/kg) while hepatic and renal biopsies were examined for histological and histochemical abnormalities. Methotrexate induced hydropic degeneration, pyknosis, sinusoidal dilatation and bile duct hyperplasia in the liver together with renal tubular degeneration, glomerular shrinkage and hyaline droplet precipitation. While propolis partially ameliorated some of the morphometric and biochemical alterations, none of the hepatic alterations induced by MTX was protected by propolis treatment. Nevertheless glomerular shrinkage and renal tubule degeneration were partially protected in animals received both MTX plus propolis. It is concluded that propolis treatment has little or no ameliorative effect in protecting the hepatic and renal tissues from MTX toxicity.

El metotrexato (MTX) es ampliamente utilizado en el tratamiento de algunas formas de cáncer, pero tiene efectos secundarios graves. El presente trabajo tuvo como objetivo investigar el papel protector del tratamiento con própoleo frente a las alteraciones inducidas por el MTX en los tejidos hepático y renal. Se expusieron conejos a MTX (0,25 mg / kg), en grupos con y sin propóleo (50 mg / kg), y se realizaron biopsias hepáticas y renales, que fueron examinadas buscando anomalías histológicas e histoquímicas. El metotrexato indujo la degeneración hidrópica, picnosis, dilatación sinusoidal e hiperplasia del conducto biliar en el hígado, junto con la degeneración tubular renal, la contracción glomerular y la precipitación hialina. Mientras que el propóleo parcialmente mejoró algunas de las alteraciones morfométricas y bioquímicas, ninguna de las alteraciones hepáticas inducidas por MTX fue protegido por el tratamiento con propóleo. Sin embargo, la contracción glomerular y la degeneración de los túbulos renales fueron parcialmente protegidos en animales que recibieron MTX más propóleo. Se concluye que el tratamiento con propóleo tiene poco o ningún efecto mejorador en la protección de los tejidos hepáticos y renales sometidos a la toxicidad de MTX.

Analysis of genetic polymorphism and biochemical characterization of a functionally decreased variant in prostacyclin synthase gene (CYP8A1) in humans.

Prostacyclin synthase (CYP8A1) is an enzyme responsible for the biosynthesis of prostacyclin (PGI2) which inhibits platelet activation and exhibits anti-inflammatory effect. The objectives of this study were to identify CYP8A1 genetic variants and characterize functional consequences of CYP8A1 variants. In total, 27 variants including four previously unidentified single-nucleotide polymorphisms (SNPs) were identified by direct DNA sequencing in Koreans (n=48). Among them, CYP8A1 A447T and E314Stop were newly assigned as CYP8A1(∗)5 and CYP8A1(∗)6 by the Human Cytochrome P450 Allele Nomenclature Committee, respectively. CYP8A1(∗)5 was found in the heme binding area in three individuals as a heterozygous mutation. To investigate the functional change of CYP8A1(∗)5, CYP8A1(∗)5 and wild-type CYP8A1 protein were overexpressed in an Escherichia coli expression system and purified. Metabolism of PGH2 by the CYP8A1(∗)5 protein exhibited significantly decreased activity, resulting in a 45% decrease in Vmax and a 1.8-fold decrease in intrinsic clearance compared to the wild-type. Based on the predicted crystal structure of CYP8A1(∗)5 using the Molecular Operating Environment platform, the distance from CYP8A1 Cys441 to the heme was altered with a significantly changed binding free energy for the mutant protein. Further studies would be needed to determine the effect of CYP8A1(∗)5 on PGI2 levels in humans.

Identification of cytochrome P450s involved in the metabolism of arachidonic acid in human platelets.

Although cytochrome P450s (CYPs) have been identified in most human cells, identification of CYPs in human platelets remains poorly explored. CYP expressions in human platelets were screened by using reverse transcriptase-polymerase chain reaction and western blot analysis followed by functional assays using arachidonic acid (ARA). CYP1A1, 2U1, 2J2, 4A11, 4F2, and 5A1 were expressed as both proteins and mRNAs in platelets. Ethoxyresorufin-O-deethylase activity was observed in platelets and this activity was significantly decreased after treatment with the general P450 inhibitor SKF-525A and the CYP1A inhibitor, α-naphthoflavone (40-45%, P<0.001). Seventeen ARA metabolites were detected in ARA-treated platelets. Among these, the levels of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids were significantly decreased with the treatment of the P450 ω-hydroxylase inhibitor 17-octadecynoic acid (P<0.05-0.001). In summary, multiple ARA-metabolizing P450s were identified in human platelets. These findings may provide an important resource for understanding physiological function of platelet.

N-acetyltransferase-2 genotypes among Jordanian patients with diabetes mellitus.

BACKGROUND: There are inconsistent reports concerning N-acetyltransferase 2 (NAT2) genotypes in diabetes mellitus (DM). OBJECTIVE: The objective of the study was to explore any association between NAT2 genotypes and Type 1 and Type 2 DM in Jordanians. METHODS: 106 Type 1 and 110 Type 2 DM patients attending the "National Center for Diabetes, Endocrinology and Genetics", Amman, Jordan, were included in the study. DNA was extracted from venous blood using a commercial DNA extraction kit. NAT2 genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequency of the genotype that encodes rapid acetylation (the wild-type genotype NAT2*4/4) was similar in the two types of diabetes mellitus. Those which encode intermediate acetylation (NAT2*4/5, NAT2*4/6, and NAT2*4/7) were higher in Type 2 diabetes (0.482) compared to Type 1 diabetes (0.339), while the frequency of genotypes which encode slow acetylation (NAT2*5/5, NAT2*5/6, NAT2*5/7, NAT2*6/6, NAT2*6/7, and NAT2*7/7) were higher in Type 1 diabetes (0.547) compared to Type 2 diabetes (0.418). CONCLUSION: There is excess of genotypes encoding intermediate acetylation in Type 2 DM and an excess of slow acetylator genotypes in Type 1 DM. Furthermore, NAT2*4/6 genotype (which encodes intermediate acetylation) was more prevalent in Type 2 DM. Type 1 DM behaved similar to non-diabetic controls in regard to acetylation status.

Single nucleotide polymorphisms in SULT1A1 and SULT1A2 in a Korean population.

SULT1A1 and SULT1A2 are encoded on the same chromatid, and exhibit a 96% amino acid similarity. To screen for genetic variants in these two closely related genes, SULT1A1 and SULT1A2 were directly sequenced in 50 healthy Koreans. A total of 30 variations were identified in SULT1A1: eight in exons, thirteen in introns, and nine in the 5'-untranslated region. With regard to SULT1A2, 21 variants were identified, comprising seven in exons, five in introns, and nine in the 5'-untranslated region. Among these 51 variations, one in SULT1A1 and eight in SULT1A2 were previously unidentified, which include three coding variants (SULT1A2 R37Q, 110G>A; SULT1A2 G50S, 148G>A; SULT1A2 F286L, 3819C>A) and one null allele (SULT1A2 E217Stop, 3542G>T). Two LD blocks, major haplotype structures, and 7 haplotype-tagging SNPs were determined together for SULT1A1 and SULT1A2 as a single set. Frequencies of common functional variants were compared among ethnic groups. Since these two SULT enzymes are on the same chromatid in a parallel direction with overlapping substrate specificities, a combined analysis using LD and haplotype-tagging single-nucleotide polymorphisms (SNPs) will facilitate understanding of the variations in the sulfation reactions of a wide range of substrates, as compared with analysis of individual genes.

Effects of testosterone and 17ß-oestradiol on expression of the G protein-coupled receptor P2Y12 in megakaryocytic DAMI cells.

P2Y12 is an important G protein-coupled receptor that is involved in ADP-induced platelet aggregation, which is essential for normal haemostasis. Gender differences in the incidence of cardiovascular disease have been proposed to be linked to the effects of sex hormones on cardiovascular-related genes. We examined the influences of testosterone and 17ß-oestradiol on P2Y12 gene expression in megakaryocytic DAMI cell line. Altered levels of P2Y12 mRNA, protein and the cAMP-dependent vasodilator-stimulated phosphoprotein-Ser157 (VASP-Ser157) phosphorylation were investigated after treatment with 17ß-oestradioal or testosterone as compared to the control groups. Quantitative real-time PCR revealed that the P2Y12 mRNA levels were increased by testosterone in a dose-dependent manner, whereas 17ß-oestrodiol had no effect on P2Y12 gene expression. Induction of the P2Y12 protein by testosterone was found in Western blots of the proteins isolated from testosterone-treated cells. Testosterone-mediated P2Y12 expression was repressed at both the transcriptional and translational levels by the anti-androgen receptor bicalutamide. Treatment with testosterone also resulted in a decrease in the level of VASP-Ser157 phosphorylation, as compared to the control group. The decrease in the level of VASP-Ser157 phosphorylation was reversed by bicalutamide. These findings suggest a novel pathway for testosterone regulation of P2Y12 expression in a megakaryocytic DAMI cell line. Further studies using primary human megakaryocytes and platelets could be necessary to know the effect of hormones on the P2Y12 expression in circulating platelets.