| 1. |
- Bylund, Johan, et al.
(författare)
-
Metabolic profiling of TRPV1 antagonists of the benzothiazole amide series : implications for in vitro genotoxicity assessment
- 2013
-
Ingår i: Xenobiotica. - : Informa UK Limited. - 0049-8254 .- 1366-5928. ; 43:2, s. 201-210
-
Tidskriftsartikel (refereegranskat)abstract
- 1. In vitro metabolic profiling and in vitro genotoxicity assessment are important aspects of the drug discovery program as they eliminate harmful compounds from further development. In standard in vitro genotoxicity testing, induced rat liver S9 is used as an exogenous bio-activation system for detecting promutagens. In this study we show that rat liver S9 is an insufficient system regarding the conversion of TRPV1 antagonists of the benzothiazole amide series into relevant in vivo metabolites. 2. Human and rat hepatocyte experiments demonstrated generation of an aryl amine metabolite that was subsequently N-acetylated. The hydrolyzed metabolites as well as the parent compound were also metabolized into glutathione (GSH) conjugates. Rat liver S9 exhibited a very low amide hydrolysis capacity and no formation of GSH conjugates when supplemented with NADPH and GSH. 3. The discrepancy in metabolic capability between hepatocytes and rat liver S9 led to confounding results in in vitro genotoxicity assessment for this chemical class as judged by the results of Ames test, mouse lymphoma assay, SOS/umu test and Comet assay in rat hepatocytes. 4. This study highlights the pivotal role that understanding the mechanism of metabolite formation has in interpreting as well as designing reliable and relevant in vitro genotoxicity experiments.
|
|
| 2. |
- Golkar, Siv Österman, et al.
(författare)
-
Intracellular deoxyribonucleotide pool imbalance and DNA damage in cells treated with hydroxyurea, an inhibitor of ribonucleotide reductase
- 2013
-
Ingår i: Mutagenesis. - : Oxford University Press (OUP). - 0267-8357 .- 1464-3804. ; 28:6, s. 653-660
-
Tidskriftsartikel (refereegranskat)abstract
- Imbalance in the nucleotide pool of mammalian cells has been shown to result in genotoxic damage. The goal of this study was to devise a sensitive, reproducible and simple method for detection of nucleotide pool changes in mammalian cells that could be used for problem-solving activities in drug development, e.g. mechanistic explanation of a positive response in a mammalian in vitro genotoxicity test. The method evaluated in this study is based on ethanol extraction of the total nucleotide pool, heat treatment and filtration, treatment with calf intestine alkaline phosphatase to convert nucleotides to nucleosides and analysis of the nucleosides by high-performance liquid chromatography with ultraviolet detection. The method was applied to measure the intracellular levels of deoxyribonucleotides in mouse lymphoma (ML) L5178Y cells treated with various concentrations of a model compound, hydroxyurea (HU), a ribonucleotide reductase inhibitor. DNA strand breakage and micronuclei formation were assessed in the same experiments. Imbalance of nucleotide pool (i.e. changes in the relative ratios between individual nucleotide pools) in HU-treated ML cells has been observed already at a concentration of 0.01 mmol/l, whereas genotoxic effects became apparent only at higher concentrations of HU (i.e. 0.25 mmol/l and higher) as indicated by formation of DNA strand breaks and micronuclei.
|
|
| 3. |
- Haghdoost, Siamak, et al.
(författare)
-
The nucleotide pool is a significant target for oxidative stress
- 2006
-
Ingår i: Free Radical Biology & Medicine. - : Elsevier Inc.. - 0891-5849 .- 1873-4596. ; 41:4, s. 620-626
-
Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is considered to be one of the most important phenomena involved in the process of aging and age-related diseases. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) has been frequently used as a marker for oxidative stress. However, the origin of extracellular 8-oxo-dG is not well understood. The aim of this work was to investigate the nucleotide pool and the role of the human mutT homologue protein (hMTH1) in the appearance of extracellular 8-oxo-dG in a cellular model system. For this purpose we used primary human fibroblast cells, which were transfected by siRNAs homologous to hMTH1. Extracellular 8-oxo-dG in cell culture media after exposure of the cells to ionizing radiation was measured as enzyme-linked immunosorbent assay reactivity. Our results demonstrate the profound effect of both hMTH1 expression and nucleotide pool size on the cellular excretion of 8-oxo-dG, suggesting that the nucleotide pool is a significant target for the formation of extracellular 8-oxo-dG.
|
|
| 4. |
- Olsson, Gunilla, et al.
(författare)
-
Transient delay of radiation-induced apoptosis by phorbol acetate
- 2016
-
Ingår i: Radiation and Environmental Biophysics. - : Springer Science and Business Media LLC. - 0301-634X .- 1432-2099. ; 55:1, s. 95-102
-
Tidskriftsartikel (refereegranskat)abstract
- The mechanisms of interference of a model tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) with radiation-induced apoptosis in human peripheral lymphocytes have been investigated. The cells were treated with TPA under various conditions and thereafter exposed to a single lethal dose of gamma radiation. Morphological and biochemical changes characteristic of apoptosis were followed up to 72 h of post-irradiation time. Acute exposure to low concentration of TPA resulted in delay in the onset of radiation-induced apoptosis (determined as morphological changes and rate of mitochondrial demise) by 24-48 h as compared to the irradiated, sham TPA-treated cells. The time course of this delay correlated well with confinement of the p53 protein to the cytoplasm and increase in bcl-2 levels at the nuclear periphery of irradiated cells. Our results indicate that confinement of p53 in the cytoplasm is one of the potential mechanisms by which TPA interferes with the process of radiation-induced apoptosis in human lymphocytes.
|
|
| 5. |
- Salmela, Elina, et al.
(författare)
-
Swedish population substructure revealed by genome-wide single nucleotide polymorphism data
- 2011
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:2, s. e16747-
-
Tidskriftsartikel (refereegranskat)abstract
- The use of genome-wide single nucleotide polymorphism (SNP) data has recently proven useful in the study of human population structure. We have studied the internal genetic structure of the Swedish population using more than 350,000 SNPs from 1525 Swedes from all over the country genotyped on the Illumina HumanHap550 array. We have also compared them to 3212 worldwide reference samples, including Finns, northern Germans, British and Russians, based on the more than 29,000 SNPs that overlap between the Illumina and Affymetrix 250K Sty arrays. The Swedes--especially southern Swedes--were genetically close to the Germans and British, while their genetic distance to Finns was substantially longer. The overall structure within Sweden appeared clinal, and the substructure in the southern and middle parts was subtle. In contrast, the northern part of Sweden, Norrland, exhibited pronounced genetic differences both within the area and relative to the rest of the country. These distinctive genetic features of Norrland probably result mainly from isolation by distance and genetic drift caused by low population density. The internal structure within Sweden (F(ST) = 0.0005 between provinces) was stronger than that in many Central European populations, although smaller than what has been observed for instance in Finland; importantly, it is of the magnitude that may hamper association studies with a moderate number of markers if cases and controls are not properly matched geographically. Overall, our results underline the potential of genome-wide data in analyzing substructure in populations that might otherwise appear relatively homogeneous, such as the Swedes.
|
|
| 6. |
- Stevens, Kristen N, et al.
(författare)
-
19p13.1 is a triple negative-specific breast cancer susceptibility locus
- 2012
-
Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 72, s. 1795-
-
Tidskriftsartikel (refereegranskat)abstract
- The 19p13.1 breast cancer susceptibility locus is a modifier of breast cancer risk in BRCA1 mutation carriers and is also associated with risk of ovarian cancer. Here we investigated 19p13.1 variation and risk of breast cancer subtypes, defined by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) status, using 48,869 breast cancer cases and 49,787 controls from the Breast Cancer Association Consortium (BCAC). Variants from 19p13.1 were not associated with breast cancer overall or with ER-positive breast cancer but were significantly associated with ER-negative breast cancer risk [rs8170 Odds Ratio (OR)=1.10, 95% Confidence Interval (CI) 1.05 - 1.15, p=3.49 x 10-5] and triple negative (TN) (ER, PR and HER2 negative) breast cancer [rs8170 OR=1.22, 95% CI 1.13 - 1.31, p=2.22 x 10-7]. However, rs8170 was no longer associated with ER-negative breast cancer risk when TN cases were excluded [OR=0.98, 95% CI 0.89 - 1.07, p=0.62]. In addition, a combined analysis of TN cases from BCAC and the Triple Negative Breast Cancer Consortium (TNBCC) (n=3,566) identified a genome-wide significant association between rs8170 and TN breast cancer risk [OR=1.25, 95% CI 1.18 - 1.33, p=3.31 x 10-13]. Thus, 19p13.1 is the first triple negative-specific breast cancer risk locus and the first locus specific to a histological subtype defined by ER, PR, and HER2 to be identified. These findings provide convincing evidence that genetic susceptibility to breast cancer varies by tumor subtype and that triple negative tumors and other subtypes likely arise through distinct etiologic pathways.
|
|
| 7. |
- Åsgård, Rikard, et al.
(författare)
-
Evidence for different mechanisms of action behind the mutagenic effects of 4-NOPD and OPD : the role of DNA damage, oxidative stress and an imbalanced nucleotide pool
- 2013
-
Ingår i: Mutagenesis. - : Oxford University Press. - 0267-8357 .- 1464-3804. ; 28:6, s. 637-644
-
Tidskriftsartikel (refereegranskat)abstract
- The mutagenicity of 4-nitro-o-phenylenediamine (4-NOPD) and o-phenylenediamine (OPD) was compared using the Mouse Lymphoma Assay (MLA) with or without metabolic activation (S9). As expected, OPD was found to be a more potent mutagen than 4-NOPD. To evaluate possible mechanisms behind their mutagenic effects, the following end points were also monitored in cells that had been exposed to similar concentrations of the compounds as in the MLA: general DNA damage (using a standard protocol for the Comet assay); oxidative DNA damage (using a modified procedure for the Comet assay in combination with the enzyme hOGG1); reactive oxygen species (ROS; using the CM-H(2)DCFDA assay); and the balance of the nucleotide pool (measured after conversion to the corresponding nucleosides dC, dT, dG and dA using high-performance liquid chromatography). Both compounds increased the level of general DNA damage. Again, OPD was found to be more potent than 4-NOPD (which only increased the level of general DNA damage in the presence of S9). Although less obvious for OPD, both compounds increased the level of oxidative DNA damage. However, an increase in intracellular ROS was only observed in cells exposed to 4-NOPD, both with and without S9 (which in itself induced oxidative stress). Both compounds decreased the concentrations of dA, dT and dC. A striking effect of OPD was the sharp reduction of dA observed already at very low concentration, both with and without S9 (which in itself affected the precursor pool). Taken together, our results indicate that indirect effects on DNA, possibly related to an unbalanced nucleotide pool, mediate the mutagenicity and DNA-damaging effects of 4-NOPD and OPD to a large extent. Although induction of intracellular oxidative stress seems to be a possible mechanism behind the genotoxicity of 4-NOPD, this pathway seems to be of less importance for the more potent mutagen OPD.
|
|
