| 1. |
- Ishihara, M., et al.
(author)
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Quantitative structure-cytotoxicity relationship analysis of phenoxazine derivatives by semiempirical molecular-orbital method
- 2007
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In: Anticancer Research. - 0250-7005 .- 1791-7530. ; 27:6B, s. 4053-4057
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Journal article (peer-reviewed)abstract
- A semiempirical molecular-orbital method (CAChe) was applied to delineate the relationship between the cytotoxicity (evaluated by 50% cytotoxic concentration, CC50) of fifteen phenoxazine derivatives and eleven physical parameters (descriptors). Most of the phenoxazine derivatives had extended and planar structure. The cytotoxic activity of phenoxazines against the human oral squamous cell carcinoma HSC-2 and HSC-4 cells correlated to electron affinity, absolute hardness (eta), absolute electron negativity (chi) and octanol-water distribution coefficient (log-P). However, only log-P was correlated to CC50 in the HSC-3 cells, whereas only heat of formation and log-P were correlated to CC50 in the human promyelocytic leukemia HL-60 cells. The cytotoxic activity of the phenoxazine derivatives became maximum at the log-P=5.9. Their cytotoxicity strongly depended on the chi value, but not on the eta value. Compounds with relatively higher cytotoxicity showed higher chi value (chi > 5.28), whereas compounds with relatively lower cytotoxicity showed lower chi value (chi < 4.27). These data suggest that appropriate chemical descriptors should be selected to estimate the cytotoxicity of phenoxazines, depending on the target cells.
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| 2. |
- Karlsson, Isabella, et al.
(author)
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Peptide Reactivity of Isothiocyanates - Implications for Skin Allergy
- 2016
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Journal article (peer-reviewed)abstract
- Skin allergy is a chronic condition that affects about 20% of the population of the western world. This disease is caused by small reactive compounds, haptens, able to penetrate into the epidermis and modify endogenous proteins, thereby triggering an immunogenic reaction. Phenyl isothiocyanate (PITC) and ethyl isothiocyanate (EITC) have been suggested to be responsible for allergic skin reactions to chloroprene rubber, the main constituent of wetsuits, orthopedic braces, and many types of sports gear. In the present work we have studied the reactivity of the isothiocyanates PITC, EITC, and tetramethylrhodamine-6-isothiocyanate (6-TRITC) toward peptides under aqueous conditions at physiological pH to gain information about the types of immunogenic complexes these compounds may form in the skin. We found that all three compounds reacted quickly with cysteine moieties. For PITC and 6-TRITC the cysteine adducts decomposed over time, while stable adducts with lysine were formed. These experimental findings were verified by DFT calculations. Our results may suggest that the latter are responsible for allergic reactions to isothiocyanates. The initial adduct formation with cysteine residues may still be of great importance as it prevents hydrolysis and facilitates the transport of isothiocyanates into epidermis where they can form stable immunogenic complexes with lysine-containing proteins.
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| 3. |
- Karlsson, Isabella, et al.
(author)
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The Fate of a Hapten - From the Skin to Modification of Macrophage Migration Inhibitory Factor (MIF) in Lymph Nodes
- 2018
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Journal article (peer-reviewed)abstract
- Skin (contact) allergy, the most prevalent form of immunotoxicity in humans, is caused by low molecular weight chemicals (haptens) that penetrate stratum corneum and modify endogenous proteins. The fate of haptens after cutaneous absorption, especially what protein(s) they react with, is largely unknown. In this study the fluorescent hapten tetramethylrhodamine isothiocyanate (TRITC) was used to identify hapten-protein conjugates in the local lymph nodes after topical application, as they play a key role in activation of the adaptive immune system. TRITC interacted with dendritic cells but also with T and B cells in the lymph nodes as shown by flow cytometry. Identification of the most abundant TRITC-modified protein in lymph nodes by tandem mass spectrometry revealed TRITC-modification of the N-terminal proline of macrophage migration inhibitory factor (MIF) - an evolutionary well-conserved protein involved in cell-mediated immunity and inflammation. This is the first time a hapten-modified protein has been identified in lymph nodes after topical administration of the hapten. Most haptens are electrophiles and can therefore modify the N-terminal proline of MIF, which has an unusually reactive amino group under physiological conditions; thus, modification of MIF by haptens may have an immunomodulating role in contact allergy as well as in other immunotoxicity reactions.
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| 4. |
- Ramzy, Ahmad G., et al.
(author)
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Investigation of diethylthiourea and ethyl isothiocyanate as potent skin allergens in chloroprene rubber
- 2015
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In: Contact Dermatitis. - : Wiley. - 0105-1873 .- 1600-0536. ; 72:3, s. 139-146
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Journal article (peer-reviewed)abstract
- BackgroundExposure to chloroprene rubber has resulted in numerous cases of allergic contact dermatitis, attributed to organic thiourea compounds used as vulcanization accelerators. However, thiourea compounds are not considered to be strong haptens.ObjectivesTo analyse common commercial chloroprene materials for their contents of diethylthiourea (DETU), dibutylthiourea (DBTU), diphenylthiourea (DPTU), and their degradation products, isothiocyanates; and to investigate the sensitization potencies of possible degradation products of the mentioned thiourea compounds.MethodsLiquid chromatography/mass spectrometry (MS) was used for quantification of organic thiourea compounds in chloroprene products, such as medical, sports and diving gear; isothiocyanates were measured by solid-phase microextraction/gas chromatography/MS. Sensitization potencies were determined with the murine local lymph node assay (LLNA).ResultsDETU was identified at concentrations of 2.7-9.4 mu g/cm(2) in all samples, whereas neither DBTU nor DPTU was detected. At 37 degrees C, degradation of DETU in the materials to ethyl isothiocyanate (EITC) was detected. EITC and ethyl isocyanate showed extreme and strong sensitization potencies, respectively, in the LLNA.ConclusionsDETU can act as a prehapten, being degraded to EITC when subjected to body temperature upon skin contact. EITC could thus be the culprit behind allergic contact dermatitis caused by chloroprene rubber.
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| 5. |
- Samuelsson, Kristin, 1979, et al.
(author)
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Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy
- 2009
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In: Contact Dermatitis. - : Wiley. - 0105-1873 .- 1600-0536. ; 61:2, s. 91-100
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Journal article (peer-reviewed)abstract
- Background The allergenic potency of a hapten is related to its skin penetration properties, but little is known about the distribution of haptens in the skin following topical application. Objectives The aim of this study was to investigate the diffusion and epidermal distribution using two-photon microscopy (TPM) of two fluorescent compounds. Methods Sensitizing capacities of fluorescein isothiocyanate (FITC) and fluorescein were investigated using the local lymph node assay. Chemical reactivity of the compounds was analysed, and their distribution in human epidermis was visualized using TPM and confocal microscopy. Also the in vitro diffusion through epidermis of FITC and fluorescein has been examined. Results FITC was classified as an extreme sensitizer, whereas fluorescein was non-sensitizing. TPM and confocal microscopy showed an accumulation of FITC in stratum corneum (SC), whereas fluorescein was more evenly distributed in epidermis. The diffusion of fluorescein through epidermis was three times higher than that of FITC. Conclusions TPM, which has never been used in this context before, is a promising tool for visualizing the distribution of fluorescent compounds of varying reactivity in intact skin. The strong allergen FITC is mainly retained in or adjacent to SC, whereas most fluorescein diffused through the epidermis.
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| 6. |
- Samuelsson, Kristin, 1979, et al.
(author)
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Diphenylthiourea, a common rubber chemical, is bioactivated to potent skin sensitizers
- 2011
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In: Chemical Research in Toxicology. - : American Chemical Society (ACS). - 0893-228X .- 1520-5010. ; 24:1, s. 35-44
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Journal article (peer-reviewed)abstract
- Diphenylthiourea (DPTU) is a known skin sensitizer commonly used as a vulcanization accelerator in the production of synthetic rubber, for example, neoprene. The versatile usage of neoprene is due to the multifaceted properties of the material; for example, it is stretchable, waterproof, and chemical- and abrasion-resistant. The wide application of neoprene has resulted in numerous case reports of dermatitis patients allergic to DPTU. The mechanism by which DPTU works as a contact allergen has not been described; thus, the aim of the present study was to investigate if DPTU is a prohapten that can be activated by skin metabolism. The metabolic activation and covalent binding of (14)C-labeled DPTU to proteins were tested using a skinlike cytochrome P450 (P450) cocktail containing the five most abundant P450s found in human skin (CYP1A1, 1B1, 2B6, 2E1, and 3A5) and human liver microsomes. The incubations were carried out in the presence or absence of the metabolite trapping agents glutathione, methoxylamine, and benzylamine. The metabolism mixtures were analyzed by LC-radiochromatography, LC-MS, and LC-MS/MS. DPTU was mainly metabolically activated to reactive sulfoxides resulting in desulfurated adducts in both enzymatic systems used. Also, phenylisothiocyanate and phenylisocyanate were found to be metabolites of DPTU. The sensitizing capacity of the substrate (DPTU) and three metabolites was tested in the murine local lymph node assay. Two out of three metabolites tested were strong skin sensitizers, whereas DPTU itself, as previously known, was negative using this mouse model. In conclusion, DPTU forms highly reactive metabolites upon bioactivation by enzymes present in the skin. These metabolites are able to induce skin sensitization and are probable causes for DPTU allergy. To increase the possibilities of diagnosing contact allergy to DPTU-containing items, we suggest that suitable metabolites of DPTU should be used for screening testing.
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| 7. |
- Samuelsson, Kristin, 1979
(author)
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Isothiocynates as skin sensitizers. Bioactivation and distribution in skin and draining lymph nodes
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Development of contact dermatitis can be caused by electrophilic compounds (haptens) or prohaptens that come into contact with the skin. A prohapten is a compound seemingly harmless at first sight that can be transformed into reactive metabolites by metabolizing enzymes in the skin to become a hapten. The perquisites for a compound to induce contact allergy are, absorption into the skin, bioactivation of prohaptens, and reaction with endogenous proteins. The hapten-protein complex will be further processed into antigens inducing an immune response. The major aims of this thesis were: to investigate the distribution of topically applied sensitizing isothiocyanates in human- and mouse skin, to localize hapten-bound proteins in lymph nodes of exposed mice, and to explore the possibility of isothiocyanates to be formed in the skin via bioactivation. In this thesis the isothiocyanate moiety was used as a reactive handle on three fluorescent compounds; fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), and rhodamine B isothiocyanate (RBITC). The non-sensitizers fluorescein and rhodamine were used as controls. The distribution of the fluorescent compounds, of varying reactivity, in skin was visualized and FITC and TRITC were mainly located in the stratum corneum (SC) whereas fluorescein was evenly distributed and rhodamine did not penetrate into the skin to any significant degree. Even though TRITC was an extremely strong sensitizer and mainly stayed in SC, the majority of the lymph node cells of TRITC-exposed mice were fluorescent. The fluorescence was found to be incorporated in cytosolic proteins and we identified high mobility group box 1 (HMGB1) as a haptenated protein. HMGB1 is a nuclear binding protein that can also be secreted. It is described as an endogenous adjuvant and is e.g. important for DC migration. Thus, this protein might play several roles in the development of allergic contact dermatitis. To rule out which role HMGB1 plays is beyond the scope of this thesis but of major interest for future investigations. Isothiocyanates can be identified as degradation products from substituted thioureas. Diphenylthiourea (DPTU) is a known skin sensitizer used as a vulcanization accelerator in the production of e.g. neoprene. The versatile usage of neoprene is due to the multifaceted properties of the material, it is e.g. stretchable, waterproof, and chemical- and abrasion-resistant. It has resulted in numerous case reports of dermatitis patients allergic to DPTU. Also, simultaneous reactions to DPTU and phenyl isothiocyanate (PITC) in patients have been reported. Hence, the ability of DPTU to form reactive metabolites by skin metabolism was investigated. Using a skin-like cytochrome P450 (CYP) cocktail, four metabolites of DPTU were identified and adducts from trapping experiments revealed that PITC was formed upon bioactivation and not only by thermal degradation of DPTU. PITC was further metabolized into phenyl isocyanate (PIC), an even stronger sensitizer than PITC. The major adducts formed are thought to be derived from sulfenic, sulfinic and sulfonic acids of DPTU. Thus, the acids of DPTU and/or PITC, and PIC might be responsible for the allergenic activity of DPTU. In conclusion, isothiocyanates are, due to the isothiocyanate moiety, potent contact allergens. After topical exposure, isothiocyanates bind to SC, but a sufficient amount enter the skin and are further transported to the draining lymph nodes able to cause sensitization. In the draining lymph nodes, different cell types are target cells, and HMGB1 was identified as a target protein. It is of future interest to trace hapten target proteins in vivo to be able to reveal the mechanisms behind the development of contact allergy more in detail. Furthermore, we have shown that isothiocyanates can be formed by metabolic activation of prohaptens by skin CYP enzymes. Knowledge regarding biotransformation of compounds is important to be able to predict allergenic activities of compounds not allergenic themselves. Using adequate metabolites for patch testing could increase the possibility of a correct diagnosis which otherwise would have been missed due to a too low concentration of the metabolite formed in the test situation.
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| 8. |
- Suzuki, F., et al.
(author)
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Tumor-specificity and type of cell death induced by phenoxazines
- 2007
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In: Anticancer Research. - 0250-7005 .- 1791-7530. ; 27:6B, s. 4233-4238
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Journal article (peer-reviewed)abstract
- Phenoxazines have shown diverse biological activities, but tumor-specific cytotoxic activity has not been investigated. A total of 24 phenoxazine derivatives (WM1-24) was investigated for their relative cytotoxicity against human tumor cell lines vs. normal cells. WM7 and WM8 showed the highest tumor-specificity index of 4.3 and 4.8, respectively. Considerable difference in drug-sensitivity was found among these tumor cell lines. Human promyelocytic leukemia HL-60 cells showed the highest sensitivity to both WM7 and WM8, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), and human gingival fibroblast (HGF), pulp cell (HPC) and periodontal ligament fibroblast (HPLF) were the most resistant. WM7 and WM8 induced little or no internucleosomal DNA fragmentation, and activated caspase-3 in HSC-2, HSC-4 and human glioblastoma T98G cells. These compounds failed to induce autophagic cell death, as judged by acridine orange and microtubule-associated protein I light chain 3 (LC3)-GFP assays. These results suggested that the higher cytotoxicity of WM7 and WM8 are derived from the positively-charged quaternary nitrogen substituents on the phenoxazine ring and the electron density of nitrogen at N12, and that inhibition of autophagy is not always coupled with apoplosis induction.
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