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Photopatch testing of 1155 patients: results of the U.K. multicentre photopatch study group.

BACKGROUND: Photoallergic contact dermatitis can be difficult to diagnose if not appropriately investigated. Currently, the most common U.K. photoallergens appear to be sunscreen chemicals. The investigation of choice is photopatch testing (PPT), which is probably underused. In part, this is due to differences in methodology and results interpretation. OBJECTIVES: To conduct PPT using a group of sunscreen chemicals, defined indications and a standardized methodology including interpretation and relevance of reactions in patients attending for investigation at 17 centres across the U.K., Ireland and the Netherlands. METHODS: Patients (n = 1155) who fulfilled the inclusion criteria were investigated with PPT using sunscreen chemicals in addition to suspected topical products. Readings were taken at 24, 48 and 72 h following standardized ultraviolet A irradiation (5 J cm(-2)). The clinical relevance of any reaction was recorded. RESULTS: Of the 1155, 130 had allergic reactions (11.3%). Of these, 51 had photoallergy (PA) (4.4%), 64 had contact allergy (CA) (5.5%), and 15 patients had combined PA and CA (1.3%). Multiple PA was seen in some. The most common photoallergen was benzophenone-3 (27 reactions; 21%). Most reactions (60%) were clinically relevant. The most common indication for testing in patients found to have PA was a history of reacting to a sunscreen (41%). The other 59% had an exposed-site dermatitis/skin problem or a photodermatosis. Some centres (n = 8) performed readings after the standard 48-h reading, and an extra 32 PA and 22 CA reactions were detected, which were not evident at 48 h. A new photoallergen (octyl triazone) was detected in two patients. CONCLUSIONS: Sunscreen PA and CA are probably equally uncommon. Most reactions, of both reaction types, were relevant clinically. A large proportion of patients (59%) found to have PA was unaware of reacting to a sunscreen chemical, suggesting that PA should be considered as an explanation in any exposed-site dermatitis. Although this study focused on reactions at 48 h postirradiation, readings performed up to 96 h, while inconvenient, add value by detecting additional relevant responses. A previously unknown photoallergen was found, highlighting the need for awareness of novel photoallergens in the marketplace. A standardized PPT method not only encourages more use of this investigation, but also facilitates comparison of results between centres and so will improve our understanding of PA.

Adolescent↗

Comparison of the photochemical behavior of four different photoactivatable probes.

The most commonly used photoaffinity labeling probes are compared, which are aryl azides, aryl diazirines, alpha-diazocarbonyls and benzophenone-derivatives. The compounds were used under identical conditions and crosslinking efficiency, influence of water, irradiation requirements, and by-products were investigated. Using the pentapeptide thymopentin (TP5) as a model system, we synthesized four analogues by solid-phase peptide synthesis and partially N-terminal modification to obtain [p-(3-trifluoromethyl)diazirinophenylalanine5]TP5, [p-benzoylphenylalanine5]TP5, 4-azidobenzoyl-TP5 and 2-diazo-3,3,3-trifluoropropionyl-TP5. The peptides were characterized by HPLC and ion-spray mass spectroscopy. Irradiation of the peptides with two different ultraviolet sources was carried out in water, n-propanol and water/n-propanol to imitate both hydrophobic and hydrophilic peptide/protein-interactions as well as the influence of the aqueous environment. Analysis of the products with HPLC, ion-spray MS, HPLC-MS and HPLC-CID-MS revealed that (Tmd)Phe is a highly potent carbene-precursor, which can be transformed easily into uniform crosslinking products by smooth photolysis. However, the electrophilic nature of the intermediate causes a high tendency to react with water molecules. The 4-azidobenzoyl group showed comparable crosslinking efficiency, but the probability to create non-uniform irradiation products (e.g. through rearrangement) is higher, whereas the reaction with water is less dominant. In contrast, Bpa was found to have an extremely low affinity to react with water, whereas prolonged UV irradiation is needed to get complete rearrangement into a variety of products. As the absorption band of alpha-diazocarbonyls at around 350 nm possesses a low extinction coefficient, 2-diazo-3,3,3-trifluoropropionyl-TP5 could not be activated at all with the optimized irradiation conditions that we have chosen for our comparative studies.

Adjuvants, Immunologic↗

Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle.

The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [alpha-32P]Bz2ATP results in covalent binding of [alpha-32P]Bz2ATP to the 450-kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146 +/- 0.087 (n = 2) was estimated. The covalent binding of [alpha-32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P = ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold. The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [alpha-32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [alpha-32P]Bz2ATP-labeled 76-kDa tryptic fragment. Digestion of either the [alpha-32P]Bz2ATP-labeled 450-kDa or the 76-kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39-kDa fragment is the precursor of the 27-kDa and 13-kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27-kDa and 13-kDa fragments of the ryanodine receptor protein.

Adenosine Triphosphate↗

Recrystallization in different sunscreen formulations after cutaneous application.

BACKGROUND: Data on the structure of sunscreens prior to and after application to the skin and the possible impact of these factors on their efficacy are still scant. AIMS: The microscopic structure of several commercial sunscreen products containing benzophenone-3 (BP-3) was analysed under a light microscope, prior to and 2 h after application of the products to the skin, and compared with various control preparations with or without BP-3. METHODS: All tested formulations were mounted on microscope slides, viewed under a light microscope and photographed. Samples were taken directly from original packages or from freshly prepared prescription formulations. Two hours after application to the skin, the samples were taken and processed for microscopy. RESULTS: In some commercial sunscreens numerous crystals were formed upon cutaneous application, whereas others contained crystals both prior to and after epicutaneous application. A single commercial product (a lamellar ambiphyl emulsion, SPF28) retained its regular structure throughout the study. Control preparations with or without BP-3 and/or octyl methoxycinnamate contained crystals after cutaneous application. CONCLUSIONS: Ingredients, most probably BP-3, in many commercial sunscreens are prone to recrystallization while on the skin which might interfere with their UV light-absorbing function.

Journal Article↗

Sensitivity to sunscreens.

The patients sensitised to sunscreen agents who attended our Contact Dermatitis Clinic between February 1985 to March 1987 have been reviewed. 15 (5%) of 280 patients tested with sunscreens had positive reactions; 3 of them were allergic to more than one agent. The most frequent contact allergens were hydroxy methoxy methyl benzophenone (Mexenone) [6], followed by isopropyl dibenzoylmethane (Eusolex 8020/8021) [5], octyl dimethyl para-aminobenzoate (Escalol 507) [2], and one reaction each to butyl methoxy dibenzoylmethane (Parsol 1789), amyl dimethyl para-aminobenzoate (Escalol 506), and ethoxy ethyl-p-methoxy cinnamate (Givtan F). Positive photopatch tests were seen with isopropyl dibenzoylmethane, butyl methoxy dibenzoylmethane, para-aminobenzoate (PABA) and ethoxy ethyl-p-methoxy cinnamate, one reaction each.

Benzoates↗

Identification of binding domains of pituitary adenylate cyclase activating polypeptide (PACAP) for its type 1 receptor by photoaffinity labeling.

Structure-function studies and photoaffinity labeling experiments were performed to identify residues and domains of PACAP involved in the interaction with PACAP receptors. For this purpose, a series of photoreactive analogues of PACAP(1-27) containing a photoreactive benzophenone (BP) residue in different peptide structural domains were utilized to analyze the interaction of PACAP(1-27) with pig PACAP type 1 receptors. Five PACAP derivatives were created with a photoreactive amino acid in the following peptide domains: either the disordered N-terminal or the helical C-terminal domain or a short loop region within the C-terminal helical domain of the peptide. Their receptor binding properties and efficiencies were tested on pig brain PACAP receptors. The results indicate the importance of the helical C-terminal domain of PACAP(1-27) for receptor binding affinity. Monoiodination of the photoreactive analogues did not change their binding affinities. Experiments with pig brain membranes demonstrated that the 125I-labeled photoreactive analogues specifically label a protein band of M(r) 66,000. The efficiency of photoreactive labeling differed for the various analogues. These findings suggest that Tyr22 and Lys15 in PACAP (1-27) are located in or close to the hormone binding site of the PACAP type 1 receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.

Amino Acid Sequence↗

Photosensitization by fenofibrate. II. In vitro phototoxicity of the major metabolites.

Fenofibric acid, the major metabolite of fenofibrate, was found to be photolabile. Its irradiation in aqueous solution gave rise to two photoproducts, whose formation involves photodecarboxylation of the dissociated acid to an aryloxy-substituted carbanion, which is directly protonated or, alternatively, undergoes a Wittig rearrangement. A comparative in vitro phototoxicity study has been carried out on the anti-hyperlipoproteinemic drug fenofibrate, its metabolites and the photoproducts of fenofibric acid. Fenofibrate, fenofibric acid and its two photoproducts were found to be active when examined by the photohemolysis test and were able to photosensitize peroxidation of linoleic acid, as evidenced by the UV monitoring of dienic hydroperoxides. In summary, the major metabolite of fenofibrate (fenofibric acid), as well as its photoproducts, are phototoxic in vitro. This behavior can be attributed to the fact that the four compounds retain the benzophenone chromophore present in fenofibrate and is indicative of free radical-mediated photosensitization. In agreement with this rationalization, the metabolites with a reduced ketone functionality exhibit no detectable in vitro phototoxicity.

Fenofibrate↗

Formation of 7,8-dihydro-8-oxoguanine in the 1,2-dioxetane-induced oxidation of calf thymus DNA: evidence for photosensitized DNA damage by thermally generated triplet ketones in the dark.

Isolated calf thymus DNA was treated with the 1,2-dioxetanes 3-acetoxymethyl-3,4,4-tri-methyl-1,2-dioxetane, 2,3-dimethylbenzofuran dioxetane, 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), 3,3,4,4-tetramethyl-1,2-dioxetane and 3,4,4-trimethyl-1,2-dioxetane (TrMD), which on thermal decomposition generate triplet-excited carbonyl products. To monitor quantitatively the formation of the mutagenic oxidation product 7,8-dihydro-8-oxoguanine (8-oxoGua), a sensitive and selective HPLC electrochemical assay was used after acidic hydrolysis (HF/pyridine) of the dioxetane-treated DNA. High yields of 8-oxoGua (up to ca 4% of the available guanine) were obtained for HTMD and TrMD. Both were investigated in detail with respect to effects of concentration, time and temperature. The oxidative reactivity of 1,2-dioxetanes was compared with several type I (benzophenone and riboflavin) and type II (methylene blue and rose bengal) photooxidants and disodium 1,4-etheno-2,3-benzodioxin-1,4-dipropionate as a chemical source of singlet oxygen. The persistence of 8-oxoGua towards oxidation by HTMD was examined in the reaction with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) and with oxidized DNA. It was shown that, indeed, 8-oxoGua is consumed in the oxidized DNA on prolonged exposure to an excess of HTMD. The reaction of 8-oxodGuo with HTMD afforded the two 4R* and 4S* diastereomers of 9-(2-deoxy-beta-D-erythropentofuranosyl)-4, 8-dihydro-4-hydroxy-8-oxoguanine as main oxidation products. Trapping experiments with tert-butanol confirmed that hydroxyl radicals are not involved, whereas the use of the triplet quenchers sodium 9,10-dibromo-anthracene-2-sulfonate and 2,3-diazabicyclo[2.2.1]hept-2-ene established that triplet-excited states are mainly responsible for the observed DNA oxidation through type I action (electron transfer chemistry). The role of singlet oxygen was tested by means of deuterium isotope effects in D2O versus H2O, but no definitive conclusion could be reached in regard to the involvement of 1O2 in these oxidations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.

The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

Actins↗

Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

Animals↗

Chemotactic Preferences and Strain Variation in the Response of Phytophthora sojae Zoospores to Host Isoflavones.

The zoospores of Phytophthora sojae are chemotactically attracted to the isoflavones genistein and daidzein that are released by soybean roots. In this study we have examined the response of P. sojae zoospores to a wide range of compounds having some structural similarity to genistein and daidzein, including isoflavones, flavones, chalcones, stilbenes, benzoins, benzoates, benzophenones, acetophenones, and coumarins. Of 59 compounds examined, 43 elicited some response. A comparison of the chemotactic responses elicited by the various compounds revealed a primary role for the phenolic 4(prm1)- and 7-hydroxyl groups on the isoflavone structure. A few compounds acted as repellents, notably methylated flavones with a hydrophobic B ring. The chemotactic response to many of the analogs was markedly different among different strains of P. sojae.

Journal Article↗

Heterotrophic nitrifiction by Arthrobacter sp.

Arthrobacter sp. isolated from sewage oxidized ammonium to hydroxylamine, a bound hydroxylamine compound, a hydroxamic acid, a substance presumed to be a primary nitro compound, nitrite, and nitrate. The concentration of free hydroxylamine-nitrogen reached 15 mug/ml. The identification of hydroxylamine was verified by mass spectrometric analysis of its benzophenone oxime derivative. The bound hydroxylamine was tentatively identified as 1-nitrosoethanol on the basis of its mass spectrum, chemical reactions, and infrared and ultraviolet spectra. Hydroxylamine formation by growing cells was relatively independent of pH, but the accumulation of nitrite was strongly favored in alkaline solutions. The formation of hydroxylamine but not nitrite was regulated by the carbon to nitrogen ratio of the medium. The hydroxamic acid was the dominant product of nitrification in iron-deficient media, but hydroxylamine, nitrite, and 1-nitrosoethanol formation was favored in iron-rich solutions. Heterotrophic nitrification by Arthrobacter sp. was not inhibited by several compounds at concentrations which totally inhibited autotrophic nitrification.

Acetates↗

Direct activation of cyclin-dependent kinase 2 by human papillomavirus E7.

Addition of human papillomavirus (HPV) E7 CDK2/cyclin A or CDK2/cyclin E, purified from either insect cells or bacteria, dramatically upregulates histone H1 kinase activity. Activation is substrate specific, with a smaller effect noted for retinoblastoma protein (Rb). The CDK2 stimulatory activity is equivalent in high-risk (HPV type 16 [HPV16] and HPV31) and low-risk (HPV6b) E7. Mutational analyses of HPV16 E7 indicate that the major activity resides in amino acids 9 to 38, spanning CR1 and CR2, and does not require casein kinase II or Rb-binding domain functions. Synthetic peptides spanning HPV16 amino acid residues 9 to 38 also activate CDK2. Peptides containing this sequence that carry biotin on the carboxy terminus, as well as a photoactivated cross-linking group (benzophenone), also activate the complex and covalently associate with the CDK2/cyclin A complex in a specific manner requiring UV. Cross-linking studies that use protein monomers detect association of the E7 peptides with cyclin A but not CDK2. Together, our results indicate a novel mechanism whereby E7 promotes HPV replication by directly altering CDK2 activity and substrate specificity.

Amino Acid Sequence↗

Photolabeling of a 150-kDa (Na + K + Cl) cotransport protein from dog kidney with a bumetanide analogue.

(Na + K + Cl) cotransport is the major mechanism of salt transport across the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop of mammalian kidney and the site of action of "loop" diuretics such as furosemide and bumetanide. We have identified a 150-kDa protein in membranes from dog kidney cortex that is photolabeled by a radiolabeled, benzophenone analogue of bumetanide, [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA). Several pieces of evidence strongly suggest that this 150-kDa protein is at least part of the (Na + K + Cl) cotransport system. 1) Photoincorporation of [3H]BSTBA into this protein is completely blocked by inclusion of 10 microM unlabeled bumetanide in the photolysis medium. 2) Photoincorporation of [3H]BSTBA into this protein shows a saturable dependence on [3H]BSTBA concentration, with a K 1/2 (approximately 0.1 microM) very similar to that for reversible [3H]BSTBA binding to kidney membranes. 3) Photolabeling of this protein by [3H]BSTBA requires the simultaneous presence of Na, K, and Cl in the photolysis medium. 4) When crude membranes from dog kidney cortex are centrifuged on sucrose density gradients, saturable [3H]bumetanide binding and photoincorporation of [3H]BSTBA in the 150-kDa region show a very similar distribution among the 15 gradient fractions collected. [3H]BSTBA is also photoincorporated into at least two lower molecular mass proteins, the largest of which is approximately 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Polyimide-polyethylene glycol block copolymers: synthesis, characterization, and initial evaluation as a biomaterial.

Block copolyimides with varying amounts of polyethylene glycol (PEG) were synthesized and characterized by copolymerization of diaminodiphenyl ether (DDE), amino terminated PEG, and benzophenone tetracarboxylic acid dianhydride (BTDA). Strong materials were obtained, with enhanced flexibility as compared to the parent DDE-BTDA polyimide homopolymer. Incorporation of PEG led to an increase in water absorption by these copolymers, and hydrophilicity was increased as reflected by a decrease in air-water-polymer contact angle. These materials supported less cell adhesion in vitro than the parent polyimide homopolymer. Short term in vivo evaluation of these copolymers showed reduced fibrous encapsulation than was observed in the absence of PEG.

Cell Adhesion↗

Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone.

PTH maintains blood calcium concentrations within the physiological range by acting on a G protein-coupled heptahelical receptor (PTH1 Rc) located primarily in cells in bone and kidney. We have undertaken a photoaffinity cross-linking approach to elucidate the nature of the bimolecular interaction of PTH with the human (h) PTH1 Rc. Specifically, we have studied the region of the receptor that interacts with the midregion of PTH-(1-34), position 13, using a benzophenone-containing photoaffinity ligand, 125I-[Nle(8,18),Lys13(epsilon-pBz2),L-2-NaI23,Arg(26,2 7),Tyr34]bPTH-(1-34)NH2 (125I-K13). Using site-directed mutagenesis in combination with biochemical analysis, we have reduced our previously identified contact domain, 17 residues in the extracellular region of the receptor (173-189), to an 8-amino acid domain (182-189). Furthermore, we have found arginine 186 to be of critical importance to the interaction of the hPTH1 Rc with 125I-K13: modification of Arg186 to either lysine or alanine does not modify receptor avidity or signal transduction by the receptor, but eliminates cross-linking to 125I-K13.

Amino Acid Sequence↗

Barium hydroxide lime turns yellow after desiccation.

Ethyl violet is added to carbon dioxide absorbents and normally serves as an indicator of absorbent exhaustion. During the course of several prior studies of anesthetic breakdown, we noted (but did not publish) that barium hydroxide lime (BL), but not soda lime, turns yellow upon desiccation. We hypothesize that ethyl violet undergoes chemical reaction to produce a yellow colorant in desiccated BL. We qualitatively studied the time course of yellow color development during desiccation of these absorbents with dry oxygen. The yellow colorant was extracted from desiccated absorbent with diethyl ether, separated with chromatography, and analyzed with proton nuclear magnetic resonance and combined gas chromatography and mass spectrometry. The yellow color develops after BL has reached nearly complete desiccation. We successfully identified that ethyl violet decomposes into the yellow colorant 4,4'-bis(diethylamino)benzophenone upon desiccation of BL. The color is not intense, is not useful for identifying low levels of absorbent desiccation, and may be difficult to see through tinted canisters. It may be possible for BL to be sufficiently desiccated to allow chemical breakdown of anesthetics, but not yet show yellow coloration. However, if yellow coloration exists, one should assume that it has become desiccated.

Anesthesiology↗

Purification and characterization of sulochrin oxidase from Penicillium frequentans.

Sulochrin oxidase, an enzyme catalyzing regio- and stereospecific phenol oxidative coupling reaction to form (+)-bisdechlorogeodin from sulochrin, was isolated from Penicillium frequentans CMI 96659. By chromatographies on DEAE-cellulose, hydroxyapatite, Phenyl-Sepharose, Mono P, Mono Q, and HPLC gel filtration columns, sulochrin oxidase was purified to apparent homogeneity. The purified enzyme had a molecular weight of 230 K as estimated by gel filtration and 110 K as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homodimer. The enzyme showed pI 4.0 and an optimum pH of 6. The enzyme activity was strongly inhibited by the copper-chelating reagent, diethyldithiocarbamate, and did not recover its full activity even after removing the inhibitor by dialysis. However, enzyme activity was fully restored by the addition of Cu2+. Thus, sulochrin oxidase is considered to be a copper protein. The enzyme showed high substrate specificity for benzophenone compounds such as sulochrin and dihydrogeodin.

Alcohol Oxidoreductases↗