Browsing by Subject "Metabolismus"

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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.
Escherichia coli O157:H7 und seine Adaption an verschiedene Wachstumsbedingungen in vitro : Untersuchung des intrazellulären Proteoms und Kohlenhydratmetabolismus
(2014) Polzin, Sabrina; Schmidt, Herbert
The aim of this study was to investigate the linkage of metabolism and pathogenicity of EHEC bacteria under certain environmental conditions in vitro. During the investigations the influence of environmental conditions could be shown on the protein as well as on the gene expression level. In experiments using the culturel media SIEM and SCEM compared to TSB as a reference, differential expression of proteins and genes were shown. For example, decreasing motility and the formation of biofilm-like substances were observed. Furthermore, differences in metabolism and expression of chaperons and stress response systems were detected. After culturing EHEC bacteria in experimental media, increased glucose and pyruvate metabolism, increased amino acid biosynthesis, as well as increased chaperon levels were found, in contrast to decreased expression levels of stress associated proteins. The experimental procedure also affected the presence of pathogenicity factors, like EspP and EspB. Notably, the environmental conditions induced the glutamic acid resistance, providing the EHEC bacteria an unhindered passage through the human gastrointestinal tract, especially the acidic environments like the stomach. Additionally, the sialic acid esterase Z1466 along with shiga toxin, was strongly expressed. This could provide an advantage over the intestinal micro flora, in respect of growth. Effects onto glucose metabolism and amino acid biosynthesis were investigated by isotopoloque profiling experiments, prepared thankworthy by Dr. rer. nat. Wolfgang Eisenreich (Munich). To conclude, stress response and expression of pathogenicity factors could be induced in vitro in the cultural media elected, and without the need of a human host. The experiment also advices the complex regulation of different processes in bacterial cells as response to external factors.
Saponin fractions from fenugreek (Trigonella foenum-graecum L.)as dietary supplements for Nile tilapia (Oreochromis niloticus L.)and common carp (Cyprinus carpio L.)
(2012) Stadtlander, Timo; Focken, Ulfert
The future role and importance of aquaculture for the world food supply is more and more recognized by the public. High quality feed for semi-intensive and intensive aquaculture operations are necessary to guarantee a future continuous growth of fish production. Nutrient utilization efficiency has in the past been augmented by addition of feed additives, mainly anti-biotics and hormones. That practice is prohibited in the European Union since 1st of January 2006 and the consumer acceptance of products produced in that way is low. Around a decade ago saponins, secondary plant metabolites, have been found to be potential alternatives for aquafeeds. Saponinmixtures derived from the South American soap bark tree Quillaja saponaria, have been found to have beneficial effects on growth, feed and nutrient utilization and on metabolism of common carp Cyprinus carpio and Nile tilapia Oreochromis niloticus. Furthermore, in one of the early experiments a sex ratio in favor of males was observed in saponin fed tilapia compared to control fish not supplemented with saponins. An influence on sex ratio of Nile tilapia would be highly desirable since industrially Nile tilapia production is only financially viable if all male populations are produced since mixed sex tilapia populations tend to reproduce uncontrollable. The production of all male tilapia populations is mainly conducted by feeding the potentially environmentally hazardous and carcinogenic synthetic hormone 17--methyltestosterone to sexually undeveloped tilapia fry. The above mentioned problems led to a joint research project with partners in Israel and Palestine in which this dissertation has been conducted. The aim of the project was the fractionation of crude saponin extract derived from fenugreek Trigonella foenum-graecum and its testing in vitro and in vivo. It was assumed that a higher biological activity could be achieved if saponin fractions instead of crude mixtures would be applied. After in vitro testing, different saponin fractions or eluates, have been tested on their effects on growth, feed and nutrient utilization, metabolism, proximate composition, gene expression of GH and IGF-1, sex ratio, reproduction and gonad histology. Most of the feeding experiments were conducted with Nile tilapia but one experiment was also conducted with carp. Three of the feeding experiments have been conducted in a system capable of measuring continuous respiration of the fish. Another experiment has been conducted at a field station at Jericho, Palestine. The experiments conducted to evaluate the influence of saponin fractions on sex ratio of undifferentiated tilapia fry have been conducted primarily in a flow-through system. The tested saponin fractions and a tested sapogenin are not suitable to produce male monosex tilapia populations. The sex ratios after supplementation of diets of mixed sexed tilapia fry did only in one case show a significantly higher proportion of males than the control. In a larger scale repetition of that experiment the previous observations could statistically not be proofed. It must be assumed to be random effects or be the result of initially biased sex ratios after stocking undifferentiated tilapia fry. An experiment in which genetically female tilapias were fed with two fenugreek saponin fractions and positive and negative controls supported that finding. A long term feeding experiment conducted at Jericho revealed no influence of the long and short term supplemented Q. saponaria saponin mixtures on sex ratio and reproduction of genetically female tilapia. A similar laboratory experiment with fenugreek saponin fractions could not be evaluated in that regard. The experiments evaluating the effects of the saponin fractions on growth, feed and nutrient utilization, gene expression of GH and IGF-1 and proximate composition revealed no significant differences. But in all experiments one fraction eluated with 60%/40% (v/v) methanol/water showed numerically improved values compared to control and other fractions. Furthermore one fraction eluated with 40%/60%/ (v/v) methanol/water gave constantly numerically inferior results of tested parameters compared to control. That supports the conclusion that the 40% methanol fraction contains saponins generally referred to as anti-nutrients. In only one experiment, conducted at Jericho, significantly higher growth was observed after long term supplementation with a Q. saponaria saponin mixture containing elevated sapogenin content. The presented data in this thesis is not supporting an application of the tested saponin fractions as environmentally friendly alternative to methyltestosterone to produce all male populations of tilapia or to inhibit uncontrollable reproduction. Additional experiments are needed to evaluate different modes of application like immersion treatments or injections since during the experimental work of this thesis all tested saponins were added to the feed. An application of the tested fenugreek saponin fractions as growth promoters yields a higher potential although the experimental results are based upon low sample sizes due to capacity restrictions of the respirometric system. Therefore a repetition under near commercial or commercial conditions must be considered.