The state of nano-sized titanium dioxide (TiO2) may affect sunscreen performance.

In the past several years, there has been a trend in the sunscreen/cosmetics industry to replace micron-sized titanium dioxide (TiO(2)) particles with nanoscale materials. The increased use of nanoscale TiO(2) has resulted in questions about these and other nanoproducts. This study examines the effects of using nanoscale TiO(2) on ultraviolet (UV) attenuation in simple to complex sunscreen formulations. UV light attenuation, product stability, and potential damage to the skin barrier were examined with both nanoscale and microscale TiO(2) particles. Results indicate that none of the formulations decreased the barrier function of the skin and the best UV attenuation occurs when the TiO(2) particles are stabilized with a coating and evenly distributed such as with non-agglomerated coated nanoscale materials. This indicates that nanoscale TiO(2) may have better efficacy while lacking toxicity.

UV doses of Americans.

The UV doses of Americans were never measured, but are needed for assessing the risks of UV-related health effects. We calculated these doses using a novel approach. The Environmental Protection Agency's (EPA) National Human Activity Pattern Survey (NHAPS) recorded the activity profiles of 9386 Americans over 24 months to assess their exposure to environmental pollutants, one of which is UV radiation. NHAPS used randomized telephone interviews to get their previous day's minute-by-minute activities. From NHAPS we extracted only the outdoor-daylight data of the northern and southern indoor workers (95%), stratifying by season, sex and age (0-21, 22-40, 41-59 and 60+ years) to find the average time Americans spend outdoors. Knowing the total daylight time and that while outdoors Americans are exposed to about 30% of the available solar UV (on a horizontal plane), we calculated their percent ambients. The average American's percent ambients are 2.6 and 2.5% for northern and southern females, respectively, and 3.5 and 3.6% for northern and southern males, respectively. Men over 40 years of age have the highest ambients (4%). From their ambients we calculated their annual doses using seasonal averages of UV measurements taken daily for over 2 years by EPA Brewer spectrophotometers located in four quadrants of the United States: Atlanta, GA; Boston, MA; Bozeman, MT and Riverside, CA. The average erythemal UV doses of Americans are about 25,000 J/m2/year, 22,000 for females and 28,000 for males, or 33,000 J/m2/year including a conservative continental U.S. vacation (7800 J/m2). Thus, we can now assess the risks of UV-related health effects for Americans.

UV doses of American children and adolescents.

The ultraviolet (UV) doses of American young adults were never measured, but are needed for assessing UV-related health risks. These doses were calculated using a novel approach. The National Human Activity Pattern Survey recorded the daily minute-by-minute activities of about 2000 young adults (0-19 years) over the course of 2 years to assess their exposure to environmental pollutants. From that survey, only the outdoor daylight data of northern and southern girls and boys were extracted and stratified by season and age to find the time American children (0-5 and 6-12 years) and adolescents (13-19 years) spend outside. They spend about 10% of the day outdoors, but only get about 30% of the available terrestrial UV radiation (on a horizontal plane). American children have about the same percent personal ambients as adults (3.1%), 2.8% for girls and 3.4% for boys. Adolescents have the lowest personal ambients (2.6%), 2.1% for girls and 3.1% for boys. To get their UV doses, their percent ambients are multiplied by the total available terrestrial UV. Excluding vacation, the erythemally weighted UV doses for American children are 25 kJ/m2/year, 23 for girls and 28 for boys. Adolescents get the lowest UV exposure of any group, 21 kJ/m2/year, 18 for girls and 24 for boys. Young adult northern girls get 18 kJ/m2/year and boys get 21 kJ/m2/year, whereas southern girls get 24 kJ/m2/year and boys get 31 kJ/m2/year. The youngest children (0-5 years) get slightly higher summer doses. Thus, we can now assess the UV-related health risks for American children and adolescents.

Light and death: photons and apoptosis.

Phototherapies like photodynamic therapy (PDT), UVA1, UVB, and PUVA treat skin diseases. These phototherapies work because they alter cytokine profiles, change immune cytotoxicity in the skin, and directly kill diseased cells by apoptosis. Apoptosis is a term that only describes the morphologic changes a cell undergoes during this mode of cell death. The terms "immediate", "intermediate", and "delayed" apoptosis segregate the different apoptotic mechanisms into three kinetic categories, whereas the terms preprogrammed cell death (pre-PCD) and programmed cell death (PCD) describe the underlying mechanisms. Immediate apoptosis (T< or =0.5 h post-exposure) is triggered by singlet-oxygen damage that opens the mitochondrial megachannel, which can be mediated by PDT or UVA1 radiation. It is a pre-PCD mechanism of apoptosis, i.e., protein synthesis is not required post-insult, because all the necessary components are constitutively synthesized and only need to be activated. Intermediate apoptosis (T< or =4 h>0.5 h) is initiated by receptor cross-linking on the plasma membrane, which can be achieved using high doses of UVB or UVC radiation. It is also a pre-PCD mechanism. Delayed apoptosis (T>4 h) is induced by DNA damage that can be caused by X-rays, PUVA, UVC, UVB, UVA, and PDT. It is a PCD mechanism of apoptosis, i.e., protein synthesis is required post-insult. These three apoptotic mechanisms each access one of two "points-of-no-return" located on the mitochondrial membrane, which activate different, but not mutually exclusive, final pathways of apoptosis. This review discusses the latest findings on these apoptotic mechanisms and their implications in phototherapies.

UVA1 radiation triggers two different final apoptotic pathways.

Because ultraviolet-A1 (UVA1; 340-400 nm) radiation is used therapeutically, this in vitro study addressed the question "how does it work?" To begin addressing this question, UVA1 radiation was first established to reduce the survival of transformed T and B lymphocytes in a linear dose-dependent manner using clonogenic reproductive assays, and that cell death occurs by apoptosis using transmission electron microscopy, Annexin V, and flow cytometry. The primary mechanism was determined to be immediate pre-programmed cell death, an apoptotic mechanism that does not require protein synthesis post-insult, by quantifying the apoptotic cells over time in the absence or presence of a translation inhibitor. To explore how UVA1 radiation induces immediate pre-programmed cell death apoptosis, reactive oxygen species and mitochondrial activity were altered during exposure using a variety of agents, while a specific fluorescent probe, 5,5',6,6'tetrachloro- 1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide, was used to examine mitochondrial transmembrane depolarization. To show that UVA1 mediates singlet-oxygen damage to the mitochondrial membranes, X-rays, UVB (290-320 nm), 8-methoxypsoralen and UVA, vitamin K3, anti-Fas antibody, and blocking antibody were the negative controls, while rose bengal or protoporphyrin IX with visible light were the positive controls. Cyclosporine A, which inhibits the mitochondrial megapore from opening, was used with singlet-oxygen and superoxide-anion generators to distinguish between the two final apoptotic pathways. The collective results show that UVA1 radiation primarily mediates singlet-oxygen damage triggering immediate pre-programmed cell death apoptosis (T < 20 min) by immediately opening the cyclosporine A-sensitive ("S" site) mitochondrial megapore, while superoxide anions initiate another cyclosporine A-insensitive ("P" site) final apoptotic pathway.

Preprogrammed and programmed cell death mechanisms of apoptosis: UV-induced immediate and delayed apoptosis.

Equitoxic doses (10% clonogenic survival) of UV radiation (UVR) from the three waveband regions, i.e. UVA1 (340-400 nm), UVB (290-320 nm) and UVC (200-290 nm), were shown to induce immediate or delayed apoptosis in L5178Y-R murine lymphoma cells. Membrane and DNA damage were shown to be the most probable initiators of UVA1-induced immediate or UVR-induced delayed apoptosis, respectively. These UV-induced apoptotic processes appeared to utilize two different "core" biochemical mechanisms; however, one core mechanism could be initiated at two distinct sites (e.g. membrane or DNA) and result in disparate kinetics. In an attempt to resolve this mechanistic issue, the dependence on macromolecular synthesis of each UV-induced apoptotic mechanism was investigated. In the absence of UVR, inhibition of either transcription (actinomycin D) or translation (cycloheximide) induced apoptosis in a concentration- and time-dependent manner. These results suggest that an apoptotic mechanism exists that does not require macromolecular synthesis postinsult (constitutive). The UVR data demonstrate that UVA1-induced immediate apoptosis utilizes this constitutive mechanism (preprogrammed), while UVR-induced delayed apoptosis utilizes the well-known inducible mechanism (programmed). Therefore, there are two different core biochemical mechanisms of apoptotic death available to each cell: preprogrammed (constitutive) and programmed (inducible) cell death.

Spectral dependence of UV-induced immediate and delayed apoptosis: the role of membrane and DNA damage.

The phototoxicity of each waveband region of UV radiation (UVR), i.e., UVA (320-400 nm), UVB (290-320 nm) and UVC (200-290 nm), was correlated with an apoptotic mechanism using equilethal doses (10% survival) on murine lymphoma L5178Y-R cells. Apoptosis was qualitatively monitored for DNA "ladder" formation (multiples of 200 base pair units) using agarose gel electrophoresis, while the percentages of apoptotic and membrane-permeabilized cells were quantified over a postexposure time course using flow cytometry. The UVA1 radiation (340-400 nm) induced both an immediate (< 4 h) and a delayed (> 20 h) apoptotic mechanism, while UVB or UVC radiation induced only the delayed mechanism. The role of membrane damage was examined using a lipophilic free-radical scavenger, vitamin E. Immediate apoptosis and membrane permeability increased in a UVA1 dose-dependent manner, both of which were reduced by vitamin E. However, vitamin E had little effect on UVR-induced delayed apoptosis. In contrast, the DNA damaging agents 2,4- and 2,6-diaminotoluene exclusively induced delayed apoptosis. Thus, immediate apoptosis can be initiated by UVA1-induced membrane damage, while delayed apoptosis can be initiated by DNA damage. Moreover, the results suggest that immediate and delayed apoptosis are two independent mechanisms that exist beyond the realm of photobiology.

Immediate and delayed apoptotic cell death mechanisms: UVA versus UVB and UVC radiation.

The mechanism of cell death induced by the different waveband regions of ultraviolet radiation (UVR), i.e., UVA1 (340-400 nm), UVB (290-320 nm) and UVC (200-290 nm) was investigated, using equilethal doses (90% reproductive death) on L5178Y-R murine lymphoma cells. To distinguish between necrosis and apoptosis, the following endpoints were monitored over time using flow cytometry and transmission electron microscopy: percentage of remaining cells, membrane permeabilized cells, dead cells, apoptotic cells, and ultrastructural changes. All waveband regions of UVR were found to cause apoptosis as opposed to necrosis. However, UVA1-induced immediate (0-4 h) apoptosis, while UVB- or UVC-induced delayed apoptosis (<34 h). Moreover, the membrane permeability changes that only result from exposure to UVA1 radiation, especially to red blood cells, suggests that the immediate apoptotic mechanism involves membrane damage. Therefore, the results suggest that there are three death mechanisms available to one cell type: necrosis, immediate apoptosis, and delayed apoptosis (or programmed cell death).

Non-nuclear damage and cell lysis are induced by UVA, but not UVB or UVC, radiation in three strains of L5178Y cells.

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340-400 nm, UVASUN 2000 lamp), (2) UVA+UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (G15T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1-15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m2 for the UVASUN lamp, 75 to 390 J/m2 for the FS20 lamp and 3.8 to 17.2 J/m2 for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible+IR components (> 400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.

Long-wavelength UVA radiation induces oxidative stress, cytoskeletal damage and hemolysis.

We investigated the ability of the different wavelength regions of UV radiation, UVA (320-400 nm), UVB (290-320 nm) and UVC (200-290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (> 340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB- and UVC-irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA1-exposed samples. The decrease in RCN was dependent on dose (0-1625 kJ/m2), time (0-78 h postirradiation) and cell density (10(6)-10(7) cells/mL).. Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy (200-700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.

Structural organization of the multienzyme complex of mammalian aminoacyl-tRNA synthetases.

The multienzyme complexes of mammalian aminoacyl-tRNA synthetases were purified from rat liver, rabbit liver, and rabbit reticulocytes according to the procedure slightly modified from Kellermann et al. [Kellermann, O., Brevet, A., Tonetti, H., & Waller, J.-P. (1979) Eur. J. Biochem. 99, 541-550]. Three forms of the synthetase complex with slightly different protein compositions were identified, suggesting a microheterogeneity of the synthetase complex. The hydrodynamic properties and the protein composition of the purified complexes were determined. The electron micrographs of the complex showed mostly amorphous particles and some hollow rings with an outer diameter of 164 A and an inner diameter of 42 A. The predicted hydrodynamic properties of several models of the complex were calculated. The properties of a ring model appear to best fit with those of the synthetase complex.

Mammalian high molecular weight and monomeric forms of valyl-tRNA synthetase.

Valyl-tRNA synthetase from rat liver sediments at 15.5 S with a Stokes radius of 90 A, corresponding to a native molecular weight of 585,000. Purification of valyl-tRNA synthetase to homogeneity by a combination of conventional and affinity column chromatography yields a fully active monomeric form of valyl-tRNA synthetase with a sedimentation coefficient of 7.7 S and a Stokes radius of 45 A. The subunit molecular weight of the monomeric valyl-tRNA synthetase is 140,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the presence of 400 mM KCl, the purified monomeric valyl-tRNA synthetase associates to a high molecular weight form. The high molecular weight valyl-tRNA synthetase in the homogenate can be readily converted to the monomeric form by controlled trypsinization. The kinetic parameters of the two forms are nearly identical. The results suggest that the high molecular weight valyl-tRNA synthetase is a homotypic tetramer and converts to the monomeric valyl-tRNA synthetase after the cleavage of a small peptide.