Browsing by Subject "Leber"

Now showing 1 - 3 of 3

Bioinformatische Analyse und funktionelle Charakterisierung von strukturellen Genvarianten in ADME-Genen in humaner Leber
(2016) Tremmel, Roman; Zanger, Ulrich M.
Pharmacogenetics is the study about inter-individual genetic variation that influences the response to drugs and other xenobiotics. A major part of this variation is due to hepatic drug metabolism with enzymes, transporters and receptors involved in the ab-sorption, distribution, metabolism and the excretion of drugs, xenobiotics and endoge-nous substances and collectively defined as ADME-genes. Genetic factors along with environmental and endogenous factors, including gender, age, inflammation processes and others are known to influence the expression and activity of ADME-genes. These influences can affect drug response, side effects or toxicity. According to newly published data, the human genome of any subject differs from a reference genome at 4.1 to 5.0 million positions. More than 99.9% of these differences are single nucleotide polymorphisms (SNP) or short insertions or deletions. Further-more, a person carries up to 2,500 structural variants, including copy number variations (CNV) affecting ~20 million bases (1000 Genomes Project Consortium et al., 2015). Thus structural variants affect more bases than SNPs. Per definition the CNVs are du-plicated or deleted DNA segments greater than 1kb and it was shown that they cover at least 12-30% of the human genome. Genome-wide studies investigating the function-ality of CNVs in the fruit fly, the mouse and in humans showed that there are genes whose expression is clearly affected by CNVs (dosage-sensitve), but also genes show-ing lower expression with increased copy number (dosage reversed) or genes without any expression alterations despite different copy number (dosage-insensitive). A prominent example of CNVs influencing drug metabolism is the phase I gene CYP2D6. Carriers of reduced or amplified gene copies show significantly altered ex-pression and enzyme activity levels and also a different drug metabolism of substrates like codeine (opioid) or tamoxifen (selective estrogen receptor antagonist) in compari-son to carriers with normal copy status of two. Genotyping of CYP2D6 gene copy num-ber may thus help to adjust drug dosage in a genotype dependent manner. In this work I investigated if further ADME-genes are affected by CNVs and if these variants have a functional impact on the expression phenotype and drug metabolism. The distribution of CNVs in the most important ADME-genes (n=340) was investigated in three independent cohorts using CNV data in a public accessible database of ge-nomic variants (DGV; dgv.tcag.ca), processed SNP microarray data of paired samples of healthy (n=269) and tumor (n=351) liver tissue of the TCGA project (http://cancergenome.nih.gov/) and ADME-panel based exon next generation sequenc-ing (NGS) applied on 150 well documented human liver samples of an in-house cohort (IKP148). For the NGS data analysis a method was developed and optimized to esti-mate the relative copy number of the ADME genes or every single exon via the read depth. The results were validated using qPCR with specific TaqMan assays. RNA-sequencing data of 50 healthy TCGA liver samples, and normalised expression data from microarray experiments applied to lymphoblastoid cell lines (LCL) from the HapMap samples and the 150 human liver samples (IKP148) were used to analyse the association between CNVs and the mRNA expression. Furthermore, in the IKP148 liver samples protein and enzymatic activity levels were available or measured using West-ernBlot and mass spectrometry for selected ADME-genes. All pharmacologically important CNVs of phase I and phase II genes, including CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1 and UGT2B17 could be confirmed in all datasets. CNVs which were known, but so far not functionally assessed were found in the phase I and II genes CES1, CYP2E1, CYP21A2, UGT2B15 and UGT2B28. In this work rare CNVs (<1%) were mainly found for transporters like ABCA2, SLC2A4 and SLC47A1. The analysis of the read depth in the IKP148 samples data revealed hybrid genes for CYP2A6 and CYP2D6 with their pseudogenes and allowed a fine mapping of the different alleles. The functional analysis further confirmed the positive association between CNVs and the mRNA expression of CYP2A6, CYP2D6, GSTM1, GSTT1, SULT1A1 and UGT2B17 in all three cohorts. The combination of all data from the NGS project in the IKP148 liver subjects, including SNP and CNV genotypes showed that 11% and 53% of the variability of CYP2A6 and CYP2D6 enzyme activity were explained by the genetic factors. In contrast the mRNA expression of the genes CES1 and CYP2E1 was not dependent of the CNV pattern in healthy liver tissue (IKP148 and TCGA) and lymphoblastoid cell lines. A detailed analysis of the protein and enzyme activity levels (chlorzoxazone-6-hydroxylation) of CYP2E1 confirmed the dosage-insensitivity in the IKP148 liver sub-jects. The dosage compensation can be principally explained by different mechanisms and could be tissue or tumor specific. Furthermore, CNV-linked genetic variants, altered miRNA regulation, incomplete inclusion of regulatory elements or coding sequences, hybridgenes, monoallelic expression, feedback loops or epigenetics could be factors which mask the CNV effect. In this work a haplotype analysis of the CYP2E1 region identified SNPs which were linked to the duplication and a reduced expression phenotype in persons with European ancestry. Using in silico prediction tools we found a relation of one of the linked SNPs in the 3’UTR with additional predicted miRNA bind-ing sites potentially regulating additional CYP2E1 gene copies. The CNV influence on the mRNA expression of the genes CYP21A2, UGT2B25 and UGT2B28 was inconsistent. Although CYP21A2 deletions were associated with a de-creased expression, gene duplications showed normal expression levels compared to samples with two copies. A significant influence of UGT2B28 CNVs was found in LCLs but not in human liver samples (IKP148 and TCGA). In total 7 of 17, 2 of 12 and 3 of 14 ADME genes showed a significant association between expression and CNV type in the IKP148, TCGA and LCLs of the HapMap samples, respectively. In the TCGA cancer tissue nearly all ADME-genes carry CNVs and in 30% of the genes a significant correlation was observed. With cooperation partners further polymorphisms and phenotypes of SULT1A1 and CYP2E1 were analyzed. CYP2E1: In this part of the thesis factors influencing the risk of developing differentiat-ed thyroid carcinoma (DTC) were investigated. Known risk factors for the progression of DTC are genetic and environmental factors, including ionizing radiations, previous thyroid diseases, and hormone factors. It has been speculated that dietary acrylamide intake correlates with the DTC formation. The acrylamide molecule is metabolized by CYP2E1 to the reactive carcinogenic glycidamide. The enzymatic reaction is probably dependent on the CYP2E1 genotype. Together with a cooperation partner (Prof. Dr. Landi, University of Pisa, Pisa, Italy) we investigated, whether CYP2E1 variants influ-ence the DTC risk. Prof. Landi and colleagues used a case-control-cohort and a haplo-type approach and observed a significant association between a tag-SNP rs2480258 (A allele), which covers variants in intron eight and the 3’UTR, and an increased DTC risk. In the human liver samples (IKP148) the rs2480258 genotypes were assessed using an imputation analysis and it was shown that particularly the A allele of the SNP reduce significantly the mRNA and protein expression and the enzyme activity. An in silico prediction for the molecular mechanism suggested that miR570 specifically down regulates the transcripts in carriers of the A allele. These results indicated that the inter-individual CYP2E1 activity as well as acrylamide (similar to glycidamide) influences the risk for DTC. SULT1A1: Methyleugenol, a secondary metabolite present in herbs such as basil or laurel is metabolized in humans by sulfation to a reactive product which can covalently bind to DNA. The resulting DNA adducts are mutagenic and can promote carcinogene-sis. Our cooperation partner Prof. Dr. Hans-Rudolf Glatt (German Institute for Human Nutrition Potsdam-Rehbruecke (DIfE), Nuthetal, Germany) had shown, that the meth-yleugenol metabolism takes place in the liver of mice and is mainly catalyzed by the phase II enzyme SULT1A1. To investigate these facts in humans, the methyleugenol DNA-adduct levels were measured by the cooperation partner in liver tissues (n=121; IKP148) using mass spectrometry. In this work the SULT1A1 protein levels were de-termined using western blot analysis and the relation between the DNA adducts as well as the SULT1A1 expression and the SULT1A1 CNVs was assessed. The SULT1A1 mRNA and protein expression were significantly correlated to the DNA adducts, e.g. higher SULT1A1 expression resulted in higher adduct levels. This emphasized the role of SULT1A1 in the in vivo metabolism in human liver samples. As mentioned above, there were individuals (IKP148) carrying one, two, three, four and five copies of SULT1A1. Deletions were found less frequent (4%) than duplications (36%). The CNVs were significantly associated with the SULT1A1 mRNA and protein expression. This result was consistent to previous studies investigating the association between SULT1A1 CNVs and enzyme activity. The methyleugenol DNA adduct levels were also significantly associated to the SULT1A1 copy number. Carriers of at least three gene copies exhibited a 2.8-fold higher DNA adduct level compared to donors carrying only one SULT1A1 gene copy. As a consequence this could mean that individuals with mul-tiple SULT1A1 copies reach faster, more often and more easily critical and ultimate adduct levels which increase the risk for developing cancer. Future studies should clari-fy whether methyleugenol intake as well as the individual SULT1A1 CNV make-up in-fluences the risk of cancer.
Geschlechtsspezifische Unterschiede in der Entstehung von alkoholbedingten Lebererkrankungen
(2010) Wagnerberger, Sabine; Bode, Christiane
Women are assumed to have a higher susceptibility to alcohol-induced liver disease (ALD) than men. Gender-related differences in food preference were described in previous studies for several populations. As certain micronutrients are reported to take influence on the development of ALD in animal experiments, the hypothesis of the present retrospective cross-sectional study was that gender-dependent (micro-) nutrient intake in patients with ALD may cause the higher susceptibility of women to this disease. In 210 patients (male: 158, female: 52) with different stages of ALD (ALD1: mild stage of liver damage; ALD2: moderately severe changes of the liver with signs of hepatic inflammation; ALD3: severely impaired liver function) and in 336 controls (male: 208, female: 128), nutrient intake was determined by a computer-guided diet history and related to the severity of ALD in dependence on the sex of the patients. No significant differences between males and females with ALD were calculated for the intake (per kg body/day) of protein, carbohydrates, fat, and the intake (per kg body/day) of most micronutrients. In females with ALD, higher intake was found for vitamin C (ALD3), calcium (ALD2), iron (ALD1 and ALD2), and zinc (ALD1), but the consumption of none of these micronutrients seems to contribute to a higher susceptibility to ALD in females. In the present study, a higher activity of ?liver-specific? enzymes and a higher DeRitis quotient was measured in female patients with ALD despite equal or lower amounts of consumed alcohol. This may indicate a higher susceptibility to the development of ALD in women. However, the data of calculated daily macro- and micronutrient intake do not suggest any explicit influence of gender-specific nutrition in the development of ALD. In a chronic setting of alcohol intake, women and female rodents are more susceptible to alcohol-induced liver disease than men and male mice. Starting from this background, the purpose of the present study was to determine if female mice are also more susceptible to acute alcohol-induced steatosis than male mice and to investigate whether this is due to alterations in hepatic lipid export. Male and female C57/Bl6-mice received one single dose of ethanol (6 g/kg) or isocaloric maltose-dextrin solution (control) intragastrically. Hepatic triglycerides, lipid accumulation, mRNA expression of microsomal triglyceride transfer protein (MTP) and apolipoprotein (Apo) B, as well as MTP activity were measured 12, 24, and 48 h after alcohol intake. In both genders, acute alcohol ingestion markedly increased hepatic lipid and triglyceride levels; however, total lipid accumulation was ~2-fold higher and more persistent in livers of female than in male mice. Fourty-eight h after ethanol treatment hepatic triglyceride concentrations in male and female ethanol-treated mice were similar to those of controls. MTP activity was significantly increased only in male mice 12 h after ethanol ingestion; whereas expression of MTP mRNA was significantly reduced in female alcohol-treated animals compared to controls at this timepoint. Expression of ApoB was also reduced only in livers of female mice after 12 h; however, differences did not reach level of significance. The results of the present study suggest that the markedly more pronounced and more prolonged susceptibility to acute alcohol-induced liver steatosis of female mice results at least partly from a gender-specific regulation of hepatic lipid export. In our experiments, the selective estrogen recepor modulator (SERM) toremifen did not protect against alcohol-induced hepatic lipid accumulation. The liver plays an important role not only in the metabolism of ethanol but also in the immune system. Lymphatic NK cells are present at an unusually high frequency among liver-resident lymphocytes (30-50 %). By producing the pro-inflammatoric and anti-fibrotic cytokine IFN-g NK cells are involved in the development of liver diseases. Results of studies of our own working group indicate a decrease of IL-12-induced IFN-g production in NK-92 cells after treatment with ethanol for 6 h. The aim of the present study was to investigate whether male (testosterone) or female (estrogen, progesterone, FSH, LH) sex hormones influence the ethanol-induced immunosuppression in NK-92 cells. Therefore, NK-92 cells were incubated with different sex hormones for 19 h and were subsequently treated with ethanol (1-3 ?) and sex hormones for 18 h. Concentrations of IFN-g were determined by ELISA. According to previous studies ethanol treatment resulted in a significant decrease of released IFN-g in comparison to NK-92 cells that were not incubated with ethanol. However, treatment with male and female sex hormones did not affect IFN-g release in NK-92 cells. The results of the present study suggest that solely ethanol treatment but not incubation with sex hormones has an immun modulating effect on NK-92 cells.
Zum Einfluß von mikrobieller Phytase und Calcium auf die Blei-, Cadmium und Zinkretention beim wachsenden Schwein
(1997) Zacharias, Bernhard; Drochner, Winfried
In the present study the influence of microbial phytase and/or calcium supplementation in rations of growing pigs (15 to 30 kg resp. 50 kg) on the retention of lead, cadmium and zinc in kidneys, liver, muscles and bones was investigated. The rations consisted of a barley-soy mixture supplemented with lead, cadmium and zinc either in the form of Pb-, Cd- and Zn-polluted barley or of CdCl2, Pb(CH3COO)2, and ZnSO4. The rations contained an average of 1,45 mg Pb, 0,78 mg Cd, and 55,7 mg Zn per kg dry matter and were given either with or without an addition of 800 U microbial phytase per kg. Compared to the reference group the addition of microbial phytase resulted in diets with a nomal calcium supply (6 g/kg) to a significant increase in the concentration of lead in the phalanx 1 and cadmium in kidneys and liver. The zinc concentration, however, only was increased in tendency in bones and liver. By augmenting the calcium concentration to 12 g/kg it was possible to avoid phytase-induced increase in the retention of lead in phalanx 1 and in the deposition of cadmium in kidneys and liver. For zinc, however, this effect of calcium could not be detected. The higher lead concentration in liver, kidneys and bones after addition of Pb(CH3COO)2 may be due to the 51 higher dietary Pb level as compared to the normally polluted barley rations. The reduced Cd-accumulation in livers and kidneys after feeding CdCl2 supplemented diets with a high calcium level may be explained by an increased formation of insoluble cadmium-calcium-phytate that cannot be hydrolysed by phytase. For the heavy metals lead, cadmium, and zinc the addition of phytase might probably result in an increased metal availability due to phytase-induced hydrolysis of the phytate complex. The effect of a calcium supply exceeding the recommended level may be explained by a reduction in the solubility of phytate which results in a decreased ability of phytate to be splitted by phytase and therefore leads to a reduced