Enhanced Dark-Field Hyperspectral Imaging and Spectral Angle Mapping for Nanomaterial Detection in Consumer Care Products and in Skin Following Dermal Exposure.

Consumer personal care products, and cosmetics containing nanomaterials (NM), are increasingly available in the Canadian market. Current Canadian regulations do not require product labeling for ingredients that are present in the nanoscale. As a result, unless voluntarily disclosed, it is unclear which products contain NM. The enhanced dark-field hyperspectral imaging (EDF-HSI) coupled with spectral angle mapping (SAM) is a recent technique that has shown much promise for detection of NM in complex matrices. In the present study, EDF-HSI was used to screen cosmetic inventories for the presence of nano silver (nAg), nano gold (nAu), and nano titanium dioxide (nTiO2). In addition, we also assessed the potential of EDF-HSI as a tool to detect NM in skin layers following application of NM products in vitro on commercially available artificial skin constructs (ASCs) and in vivo on albino hairless SKH-1 mouse skin. Spectroscopic analysis positively detected nAu (4/9 products) and nTiO2 (7/13 products), but no nAg (0/6 products) in a subset of the cosmetics. The exposure of ASCs for 24 h in a Franz diffusion cell system to a diluted cosmetic containing nTiO2 revealed penetrance of nTiO2 through the epidermal layers and was detectable in the receptor fluid. Moreover, both single and multiple applications of nTiO2 containing cosmetics on the dorsal surface of SKH-1 mice resulted in detectable levels of trace nTiO2 in the layers of the skin indicating that penetrance of NM was occurring after each application of the product. The current study demonstrates the sensitivity of EDF-HSI with SAM mapping for qualitative detection of NM present in cosmetic products per se and very low levels in complex biological matrices on which these products are applied.

Quantitative determination of singlet oxygen generated by excited state aromatic amino acids, proteins, and immunoglobulins.

Singlet oxygen quantum yields generated by excited state aromatic amino acids (tryptophan, tyrosine, phenylalanine), N-acetylated amino acids (N-acetyl-tryptophan, N-acetyl-tyrosine, N-acetyl-phenylalanine), and from selected proteins and immunoglobulins have been quantified by time-resolved phosphorescence measurements. A small, but significant, quantum yield found for proteins and immunoglobulins demonstrates that molecular oxygen can diffuse through the polypeptide matrix and can be sensitized by residues buried within the folds of protein structure.

Effect of UVC-induced damage to DNA on the intercalation of thiazole orange: a convenient reporter for DNA damage.

We report a novel method of identifying damage to DNA leading to the loss of intercalation sites. Thiazole orange (TO), an intercalating cyanine dye, fluoresces strongly when intercalated in DNA, but not free in solution. Upon UVC-induced damage to DNA, the change in TO fluorescence is greater than the change in any of the other spectral or biochemical indicators (absorbance, circular dichroism and agarose gel electrophoresis), thus providing a fast screening method to identify damage to DNA. The method is geared toward high levels of damage, such as those that may result during radiation treatment of food products.

The retinal carotenoids zeaxanthin and lutein scavenge superoxide and hydroxyl radicals: a chemiluminescence and ESR study.

PURPOSE: Carotenoids are present in many biological systems, often decreasing the formation of products of oxidative damage to biological molecules. In the macula their concentration is so high that it has been believed that the yellow color filters out damaging blue light. Recent reports that dietary lutein reduces the risk of cataract in the eye lens suggested that the antioxidant action of carotenoids, which has been inferred from decreased oxidative damage, warranted further direct investigation. METHODS: Superoxide and hydroxyl radical scavenging by lutein and zeaxanthin (retinal carotenoids), beta-carotene, lycopene, lutein esters (from marigolds), and a commercial mixture of soy carotenoids were compared to scavenging by ascorbate and ascorbyl palmitate. Radical scavenging was measured with a chemiluminescent assay (luminol) and by electron spin resonance, ESR. Inhibitory concentrations, IC(50), were determined with the luminescent assay. RESULTS: All of the carotenoids scavenged both superoxide (in ESR 30-50% at 16.7 microM) and hydroxyl radicals (in ESR 50-70% at 16.7 microM, in a luminescent assay 90-99%). CONCLUSIONS: While crocin may be unable to scavenge superoxide, some of the other carotenoids do so quite effectively. The mixtures of 15,15'-cis and all-trans-carotenoids studied by ESR and luminescent assay scavenge both superoxide and hydroxyl radicals. Lycopene and beta-carotene both scavenge superoxide more effectively than the xanthophylls of the retina, zeaxanthin and lutein. All of the carotenoids examined scavenged the hydroxyl radicals more effectively than superoxide radicals. The predominant carotenoid in the fovea of the retina, zeaxanthin, scavenged hydroxyl radicals more effectively than the other retinal carotenoid, lutein. Possible mechanisms of radical scavenging by the carotenoids are discussed.

The crystal structure of a cyanobacterial water-soluble carotenoid binding protein.

Carotenoids undergo a wide range of photochemical reactions in animal, plant, and microbial systems. In photosynthetic organisms, in addition to light harvesting, they perform an essential role in protecting against light-induced damage by quenching singlet oxygen, superoxide anion radicals, or triplet-state chlorophyll. We have determined the crystal structure of a water-soluble orange carotenoid protein (OCP) isolated from the cyanobacterium Arthrospira maxima at a resolution of 2.1 A. OCP forms a homodimer with one carotenoid molecule per monomer. The carotenoid binding site is lined by a striking number of methionine residues. The structure reveals several possible ways in which the protein environment influences the spectral properties of the pigment and provides insight into how the OCP carries out its putative functions in photoprotection.

Effect of cyclodextrins on the physicochemical properties of chlorophyll a in aqueous solution.

The interactions between chlorophyll a and two beta-cyclodextrins, that have the same cavity size but different substituents, were studied in aqueous solutions. These supramolecular host-guest complexes were examined by a combination of UV/vis absorption, circular dichroism, NMR, and steady-state and time-resolved fluorescence measurements. The results indicate that all cyclodextrins solubilize the pigment mainly in monomeric form in water. The pigment forms 1:1 complexes with the heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and 1:2 complexes with the hydroxypropyl-beta-cyclodextrin. In such complexes the methyl groups of the cyclodextrin inner cavity are involved in the interaction with the pigment as evidenced by NMR measurements. We also measured the luminescence of singlet oxygen photosensitized by chlorophyll a in the inclusion complexes.

Lens alpha-crystallin and hypericin: a photophysical mechanism explains observed lens.

Determining whether alpha-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by alpha-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (1Deltag) oxygen produced from hypericin in the presence of alpha-crystallin. alpha-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern-Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to alpha-crystallin does not inhibit singlet oxygen formation by hypericin. alpha-Crystallin reacts with singlet oxygen with a rate constant of 1.3 x 10(8) M(-1) s(-1). Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.

Tuning the singlet oxygen quantum yield of near-IR-absorbing porphyrazines.

We report the quantum yields for singlet oxygen production by a series of porphyrazines (pz) of the form M[pz(An;B4-n)] (Scheme 1), where the peripheral substituent A is [S-R]2 with R = (CH2CH2O)3H, B is a fused alpha,alpha'-dialkoxybenzo group and M = 2H, Mg or Zn. These compounds show intense near-IR absorbance/emission (longest wavelength emission, approximately 830 nm). Their solubilities vary with R, whereas their optical properties do not. We show that singlet oxygen sensitization by these luminescent compounds can be "tuned" from essentially off to on by varying n and selection among M = 2H, Mg or Zn. The quantum yields vary ca 60-fold within the set of compounds studied, from phidelta = 0.007 for compound 3 to phidelta = approximately 0.4 for compound 11.