| 1. |
- Bergander, Linda, 1972-
(författare)
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Formation and metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole - a light-induced Ah-receptor ligand
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aryl hydrocarbon receptor (AhR) is a ligand dependent transcription factor ubiquitously expressed in mammalian cells. It is a genetically ancient protein mostly known for binding the extremely toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding to the AhR explains the variety of toxic responses of TCDD as well as the induction of several drug metabolizing enzymes. Induction of cytochrome P4501A1 (CYP1A1) is the most well characterized of the AhR regulated responses. The physiological functions of AhR and the endogenous ligand(s) for the receptor are under investigation but are not yet unraveled. Several tryptophan (TRP) derived indol-containing compounds have been reported to possess AhR affinity/CYP1A1 inducing capacity and TRP mediates CYP1A1 induction by UV light. The TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ) has the highest AhR affinity described so far and it causes a rapid and transient induction of the CYP1A1 gene in human cells. A number of reports on constitutive CYP1A1 activity in cultured cells is therefore most likely explained by the presence of TRP-derived AhR ligands in cell culture media. The aims of the studies were to investigate the impact of FICZ and FICZ metabolism on CYP1A1 gene regulation, to explore the metabolic fate of FICZ and to identify whether normal laboratory light could lead to formation of FICZ and thereby contribute to earlier observed CYP1A1 inducing effects by cell culture media. Metabolic studies using fractions of Aroclor-induced and non-induced rat liver and human liver as well as heterologously expressed enzymes revealed that FICZ can be efficiently metabolized by the CYP enzymes 1A1 and 1A2 and by an unknown cytosolic enzyme, to a number of hydroxylated and other oxidized metabolites. All of the hitherto identified 11 hydroxylated metabolites of FICZ are prone to conjugation reactions by glucuronosyltranferases and sulfotransferases. The metabolites formed by human enzymes are primarily sulfated. Thus, the sulfated metabolites of FICZ will be crucial in the future analyzes of FICZ formation in vivo. FICZ was identified to be formed, not only by UV illumination, but also by normal laboratory light. The constitutive CYP1A1 activity was significantly induced through the formation of several TRP related photoproducts in light-exposed medium. One of these photoproducts was identified as FICZ. Thus, the TRP photoproduct, FICZ, fits into a model in which FICZ auto-regulates the expression of induced enzymes. It is hypothesized that FICZ might function as a chemical messenger that activates AhR in response to light and might be one of several possible endogenous AhR ligands.
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| 2. |
- Oberg, Mattias, et al.
(författare)
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Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity.
- 2005
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Ingår i: Toxicol Sci. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 85:2, s. 935-43
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Tidskriftsartikel (refereegranskat)abstract
- The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.
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| 3. |
- Wincent, Emma, 1978-
(författare)
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Direct and indirect mechanisms for aryl hydrocarbon receptor activation mediated by 6-formylindolo[3,2-b]carbazole
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aryl hydrocarbon receptor (AhR) is mostly recognized for mediating the adverse effects of dioxins. In addition, endogenous activation of the AhR seems to have important biological functions. Several studies have demonstrated an activation of the receptor when no exogenous ligand was added. Furthermore, different physical stimuli such as UV irradiation, fluid shear stress, and hyperoxia have been shown to induce AhR-dependent transcriptional activity. Together these reports indicate either the presence of endogenous ligands or a non-ligand dependent activation. While the mechanisms behind such responses are still elusive, formation of tryptophan photoproducts with high AhR-affinity has been suggested to explain the activation observed after UVB irradiation. The photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed to be an endogenous signal substance, and is the focus of the present thesis. The objective of the work presented in this thesis was to further characterize the formation and metabolism of FICZ, to identify the biotransforming enzymes required for its metabolism, and subsequently to isolate FICZ-derived metabolites in human urine. The studies reveal that FICZ is an excellent substrate for CYP1 enzymes resulting in an efficient metabolism and rapid clearance of FICZ, and a reduced or abolished affinity for the AhR. The hydroxylated metabolites are in turn very good substrates for sulfo-conjugation, and monosulfated derivatives of FICZ were identified in human urine, proving the existence of FICZ in vivo. Furthermore, disturbing the CYP1-dependent metabolic clearance of FICZ efficiently attenuated the rapid depletion of intracellular levels of FICZ, and resulted in a delayed and prolonged AhR-activation. These results suggest that inhibition of degradation of FICZ provides a potent mechanism for indirect regulation of the AhR response. The high affinity and AhR activating capacity, together with its rapid clearance by AhR regulated biotransforming enzymes and presence in humans in vivo, all strengthen the hypothesis that FICZ is an endogenous ligand for the AhR and an important biological signaling molecule.
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| 4. |
- Wincent, Emma, et al.
(författare)
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The suggested physiologic aryl hydrocarbon receptor activator and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is present in humans
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:5, s. 2690-2696
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Tidskriftsartikel (refereegranskat)abstract
- Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.
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| 5. |
- Broberg Palmgren, Karin, et al.
(författare)
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Influence of genetic factors on toluene diisocyanate-related symptoms: evidence from a cross-sectional study
- 2008
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Ingår i: Environmental Health. - 1476-069X. ; 7:15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Toluene diisocyanate (TDI) is a highly reactive compound used in the production of, e. g., polyurethane foams and paints. TDI is known to cause respiratory symptoms and diseases. Because TDI causes symptoms in only a fraction of exposed workers, genetic factors may play a key role in disease susceptibility. Methods: Workers (N = 132) exposed to TDI and a non-exposed group ( N = 114) were analyzed for genotype (metabolising genes: CYP1A1*2A, CYP1A1*2B, GSTM1*O, GSTM3*B, GSTP1 1105V, GSTP1 A114V, GSTT1*O, MPO -463, NAT1*3, *4, *10, *11, *14, *15, NAT2*5, *6, *7, SULT1A1 R213H; immune-related genes: CCL5 -403, HLA-DQB1* 05, TNF-308, TNF-863) and symptoms of the eyes, upper and lower airways ( based on structured interviews). Results: For three polymorphisms: CYP1A1*2A, CYP1A1*2B, and TNF -308 there was a pattern consistent with interaction between genotype and TDI exposure status for the majority of symptoms investigated, although it did reach statistical significance only for some symptoms: among TDI-exposed workers, the CYP1A1 variant carriers had increased risk (CYP1A1*2A and eye symptoms: variant carriers OR 2.0 95% CI 0.68-6.1, p-value for interaction 0.048; CYP1A1*2B and wheeze: IV carriers OR = 12, 1.4-110, p-value for interaction 0.057). TDI-exposed individuals with TNF-308 A were protected against the majority of symptoms, but it did not reach statistical significance. In the non-exposed group, however, TNF -308 A carriers showed higher risk of the majority of symptoms ( eye symptoms: variant carriers OR = 2.8, 1.1-7.1, p-value for interaction 0.12; dry cough OR = 2.2, 0.69-7.2, p-value for interaction 0.036). Individuals with SULT1A1 213H had reduced risk both in the exposed and non-exposed groups. Other polymorphisms, showed associations to certain symptoms: among TDI-exposed, NAT1*10 carriers had a higher risk of eye symptoms and CCL5 -403 AG+AA as well as HLA-DQB1 *05 carriers displayed increased risk of symptoms of the lower airways. GSTM1, GSTM3 and GSTP1 only displayed effects on symptoms of the lower airways in the non-exposed group. Conclusion: Specific gene-TDI interactions for symptoms of the eyes and lower airways appear to exist. The results suggest different mechanisms for TDI- and non- TDI-related symptoms of the eyes and lower airways.
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| 6. |
- Lind, Monica, et al.
(författare)
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Short-term exposure to dioxin impairs bone tissue in male rats
- 2009
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Ingår i: Chemosphere. - : Elsevier BV. - 0045-6535 .- 1879-1298. ; 75:5, s. 680-684
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Tidskriftsartikel (refereegranskat)abstract
- Chronic and sub-chronic studies in rats have previously shown that dioxin-like compounds impair the bone tissue homeostasis. In the present study, tibiae and serum were analyzed to study possible effects of short term dioxin exposure on rats. Two month old (ca. 200g) male rats were injected with 50microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) kg(-1) bw and tibiae were excised 5d following the exposure. Bone composition, dimensions and strength were analyzed by pQCT and three-point bending test on tibiae. In addition, detailed bone composition was analyzed by optical emission spectroscopy (ICP-OES) and Fourier transform infrared spectrometry (FTIR). Analysis of the serum bone biomarkers procollagen type-I N-terminal propeptide (PINP) and carboxyterminal cross linking teleopeptide (CTX) were also performed. pQCT-results showed alterations in the metaphysis, with a significant decrease in trabecular bone cross-sectional area (-19%, p<0.05) and a significant increase in total bone mineral density (+7%, p<0.05) in TCDD-exposed rats. Analyses of the bones by ICP-OES and FTIR showed that bones from exposed rats had a higher relative proportion of crystalline phosphate (+13% for a1080 and +11% for a1113, p<0.05) and lower acid phosphate content (-22% for a1145, p<0.05), resembling the composition of more mature bones. Serum analysis showed that the bone formation marker PINP was decreased (-37%, p<0.05) and that the bone resorption marker CTX was increased (+14%, p<0.05) indicating a net loss of bone tissue. In conclusion, 5d of exposure to TCDD was sufficient to negatively affect bone tissue in male rats.
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| 7. |
- Vineis, Paolo, et al.
(författare)
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Evidence of gene-gene interactions in lung carcinogenesis in a large pooled analysis
- 2007
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Ingår i: Carcinogenesis. - : Oxford University Press (OUP). - 0143-3334 .- 1460-2180. ; 28:9, s. 1902-1905
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Tidskriftsartikel (refereegranskat)abstract
- To test the hypothesis of interaction among genetic variants in increasing the individual risk of cancer, we have studied the cumulative effect on lung cancer risk of variants in three metabolic genes, CYP1A1, GSTM1 and GSTT1, which are involved in the metabolism of the tobacco smoke constituents and environmental contaminants, polycyclic aromatic hydrocarbons and of other lung carcinogens. We have selected from the Genetic Susceptibility to Environmental Carcinogens pooled analysis all the studies on lung cancer conducted after 1991 in which all variants were available. The data set includes 611 cases and 870 controls. We found a cumulative effect of the combination of the a priori ' atrisk ' alleles for these genes (P for trend 0.004). The risk of lung cancer was increased with the combination of CYP1A1*2B or CYP1A1*4 alleles and the double deletion of both GSTM1 and GSTT1 up to an odds ratio (OR) of 8.25 (95% confidence interval 2.29-29.77) for the combination including CYP1A1*4; among never smokers, the latter combination was associated with an OR of 16.19 (1.90-137). Estimates did not change after adjustment by the number of cigarettes smoked and duration of smoking were consistent across ethnicities and were approximately the same for adenocarcinomas and squamous cell carcinomas. These observations from a large pooled analysis strongly suggest the existence of gene-gene interactions in lung carcinogenesis. People with rare combinations of common gene variants have a high risk of cancer and can be assimilated to subjects with highly penetrant mutations.
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