Alternative methods to evaluate the protective ability of sunscreen against photo-genotoxicity

Numerous epidemiological investigations show that sunlight is carcinogenic to humans and that the use of sunscreen may be effective in decreasing the risk of skin cancer. The biological activity of a sunscreen is evaluated by its ability to protect human skin from erythema as represented by a Sun Protection Factor (SPF). We propose that the sunscreen's protective effect against sunlight-induced genotoxicity, including mutation, should also be taken into account. In this study we examined the protective ability of sunscreens against natural sunlight and UV-induced genotoxicity in Drosophila somatic cells. We prepared three kinds of sunscreen samples, each with an SPF value of 20, 40 or 60 and compared their protective activities with commercial sunscreens. When a sunscreen of SPF 20, 40 or 60 was pasted on the plastic cover of a petri dish in which Drosophila larvae were exposed to the sun or UV lamps, genotoxicity decreased as the SPF of the sunscreen increased, relative to levels of genotoxicity observed in samples without sunscreen. However, the protective abilities of sunscreens were unexpectedly not so different from each other. To reveal the relationship between the protective activity of sunscreen and the wavelength of light with which larvae were irradiated through the sunscreen, we measured the transmittance of light through the petri dish cover on which the sunscreen was pasted. Effective protection was demonstrated by removing components of light whose wavelengths were below 315 nm. We suggest, that the measurement of anti-genotoxic activity and the determination of the wavelengths of light transmitted through the sunscreen should be an alternative method for evaluating the effectiveness of a sunscreen.     

A new method to test the effectiveness of sunscreen ingredients in a novel nano-surface skin cell mimic

Photophysical properties of sunscreens are commonly studied in solvent media, which do not mimic the skin, or in complex artificial skin systems, which are difficult to handle. In an earlier study, we showed that polystyrene nanosphere suspensions mimic the mixed polarity environment of skin cell systems. This paper presents a new method to quantify the effectiveness of sunscreens in the polystyrene nanosphere environment. This method utilizes the intrinsic UV-B fluorescence of polystyrene nanospheres. We studied three UV-B sunscreens by this new method and compared their extinction coefficients with observed values in solvent. The values follow the trend observed in solvents, but the ratio of their extinction coefficient in solvent to the value obtained by this new method is 1.3-1.8 instead of 1. This difference might be caused by the mixed polarity or the microgeometry of the nanosphere system. Regardless of the difference in the extinction coefficients, this new system can be used to test hundreds of chemicals for their sunscreening potential in a cost-effective way. One marked advantage of this new method is its ability to test both hydrophobic and hydrophilic sunscreening chemicals in the same environment. This is virtually impossible for current solvent-based models, which require different solvents for hydrophobic and hydrophilic chemicals. The new method also allows the simultaneous evaluation of a host of photophysical properties of sunscreening chemicals.     

Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposures to ultraviolet light

Abstract—The influence of photoreactivating light on the fate of UV-induced DNA damage has been measured in the epidermis of hairless mice using damage-specific endonuclease from Micrococcus luteus. Groups of mice were exposed to varying fluences of UV at 297nm or from an FS40 fluorescent sun lamp to induce UV photoproducts. The same fluence-dependent DNA damage was observed in high molecular weight epidermal DNA regardless of whether the mice were killed immediately, or maintained in the dark or under photoreactivating light for 20 h after UV. Thus, no detectable photoreactivation of UV-induced pyrimidine dimers could be demonstrated in mouse epithelial cells in vivo.     

Chronic ultraviolet B radiation-induced biochemical changes in the skin of hairless mice

Skh:HR-1 hairless mice were irradiated chronically with sub-erythemal doses of UVB radiation, and a number of biochemical parameters in the skin were determined after 6, 12, 18, and 24 wk of exposure. The parameters measured were water, collagen, elastin, and glycosaminoglycan content; collagenase and elastase levels; and Bz-Tyr-OEt (N-benzoyl-L-tyrosine ethyl ester) and BAPNA (alpha-N-benzoyl-DL-arginine-p-nitroanilide) hydrolyzing activities. Data for UVB radiation-exposed and chronological age-matched control mice were compared with respect to unit area and to unit mass of skin. On a unit area of skin basis, UVB radiation exposure increased the level of most parameters. The particular exceptions were collagen and collagenase which remained constant. On a mass of skin basis, though, there is an apparent decrease in collagen content because of the increase in the other skin components. This suggests that there is insufficient collagen in UVB radiation-exposed skin to support the increasing mass of the tissue.     

Quantitative determination of UV filters in sunscreen lotions using microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) has been applied to the separation of some UV filters (Eusolex 4360, Eusolex 6300, Eusolex OCR, Eusolex 2292, Eusolex 6007, Eusolex 9020, Eusolex HMS, Eusolex OS, and Eusolex 232) commonly used in sunscreen lotions. Use of a MEEKC buffer with a mixed surfactant system to stabilize the oil droplets and an organic modifier in the aqueous phase allowed separation of most of the selected analytes in a single run in a system fitted with a diode array detector recording three wavelengths (240 nm, 300 nm, and 380 nm) simultaneously. The microemulsion employed consisted of 2.25 g of SDS, 0.75 g of Brij 35, 6.6 g 1‐butanol, 0.8 g n‐octane, 17.5 g 2‐propanol, and 72.1 g of 10 mM borate buffer (pH 9.2). Detection limits from 0.65 to 6.0 μg/mL were obtained and the calibration plots were linear over at least one order of magnitude for all analytes. The developed method could be applied to the determination of UV filters in several sun protection products including lotions, milks, and oils. Comparison of the obtained data with those from an HPLC method described in the literature showed acceptable agreement.     

Determination of sunscreen agents in cosmetic products using microwave-assisted extraction and liquid chromatography

Microwave-assisted extraction (MAE), was used to extract sunscreen agents from cosmetic products. The extracts were analyzed by liquid chromatography (LC). The present method allows the determination of three sunscreen agents, Eusolex 2292, 4360 and 6300. The precision of the assay at 40 microg/ml of sunscreen agents ranged from 1.5 to 2.2%, and the detection limits were 2.0-4.0 ng/ml.     

Evaluation of sunscreen protection in human melanocytes exposed to UVA or UVB irradiation using the alkaline comet assay

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 microL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4 degrees C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37 degrees C were quantified using the comet assay. Tail moments (TM) (tail length x %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a chi 2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.     

Application of a quantitative carrier test to evaluate microbicides against mycobacteria

Microbicides for reprocessing heat-sensitive medical devices, such as flexible endoscopes, must be mycobactericidal to reduce the risk of nosocomial infections. Suspension test methods currently used for efficacy evaluation lack the stringency required for assessing inactivation of mycobacteria on surfaces. The quantitative carrier test method reported here is based on mycobacteria-contaminated reference carrier disks of brushed stainless steel. Each disk was contaminated with 10 microL of a suspension of Mycobacterium terrae containing a soil load. Each disk with a dried inoculum was placed in a glass or Teflon vial, and then overlaid with 50 microL of the test formulation or 50 microL saline for the control carriers. Five test and 3 control disks were used in each run. At the end of the contact time, each vial received 9.95 mL neutralizer solution with 0.1% Tween-80 to stop the reaction and perform the initial microbicide dilution. The inoculum was eluted by mixing on a Vortex mixer for 60 s, and the eluates and saline used to subsequently wash the vials and the funnels were membrane-filtered. Filters were placed on plates of Middlebrook 7H11 agar and incubated at 37 degrees C for at least 30 days before colonies were counted and log10 reductions were calculated in colony-forming units. Tests with a range of commercially available products, having claims against mycobacteria, or believed to be broad-spectrum microbicides, showed that the method gave reproducible results. Products used included oxidizing agents (sodium hypochlorite and an iodophore), a phenolic, a quaternary ammonium compound, and ortho-phthalaldehyde. This method represents a much more realistic evaluation than the currently used quantitative suspension test method for the evaluation of mycobactericidal formulations for registration and, when performed at different product concentrations, allows an assessment of any safety margin or risks in using the test formulation in the field.     

Prevention of UV radiation-induced premature skin aging in hairless mice by the novel compound Melanocin A

Repetitive exposure of the skin to UV radiation induces various harmful changes, such as thickening, wrinkle formation, inflammation and carcinogenesis. A variety of natural compounds and synthetic compounds have been studied to determine whether they can prevent UV-induced harmful effects. In this study, we investigated the effect of a novel compound, Melanocin A, which was isolated from Eupenicillium shearii F80695, on UV-induced premature skin aging. First, we studied the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT, in vitro. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated the effect of Melanocin A on UV-induced skin changes in hairless mice in vivo. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. Taken together, these results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging.     

超高效液相色谱-二极管阵列检测法同时测定化妆品中22种防晒剂

建立了一种超高效液相色谱-二极管阵列检测法同时测定不同种类化妆品中22种防晒剂的方法.样品首先加入少量四氢呋喃,涡旋、分散、混匀(蜡质化妆品可于50℃超声,以便分散完全),然后加入0.1％(v/v)甲酸乙醇溶液振摇、超声提取,稀释、过滤后经Poroshell 120 EC-C18(100 mm×4.6 mm,2.7μm...     

The feasibility of using fluorescence spectroscopy as a rapid, non-invasive method for evaluating sunscreen performance

We have carried out ex vivo studies to examine the feasibility of using fluorescence spectroscopy as an in vivo quantitative technique to assess sunscreen substantivity in terms of skin surface thickness and/or photoprotection. We found that the majority of sunscreens produced insufficient natural fluorescence and so we have attempted to increase the fluorescent signal by adding various fluorescing agents to the sunscreens. However, none of these substances is ideal; either they do not bind sufficiently strongly to sunscreen products, or their fluorescence is quenched by the active ingredients contained within sunscreens. The feasibility of using fluorescence spectroscopy for in vivo quantitative assessments of sunscreen substantivity therefore remains unproved and is dependent on a suitable fluorescent agent being found. Such an agent would have to be non-toxic, mix readily with sunscreens and be excited by visible wavelengths.     

Quadrupole-bound anions: efficacy of positive versus negative quadrupole moments

A pseudopotential method is utilized to study the critical stability of model anions formed by long-range quadrupolar molecular potentials. Results indicate that critical quadrupole moments of simple point-charge triads do not serve well as predictors of real quadrupole-bound anions of systems with negative moments.     

Protective effects of an antioxidant derived from serine and vitamin B6 on skin photoaging in hairless mice

The generation of reactive oxygen species (ROS) by ultraviolet radiation (UVR) accelerates skin aging, which is known as photoaging. Because cutaneous iron catalyzes ROS generation, sequestering iron by chelating agents is thought to be an effective approach toward preventing photoaging. Previously, N-(4-pyridoxylmethylene)-l-serine (PYSer) was designed as an antioxidant to suppress iron-catalyzed ROS generation by its iron-sequestering activity. In this study, PYSer showed protective effects against skin damage in hairless mice irradiated with ultraviolet B (UV-B). Topical application of PYSer to the skin significantly delayed and/or decreased the visible wrinkle formation induced by chronic UV-B irradiation. A histological study indicated that UV-B-induced epidermal hypertrophy and lymphocytic infiltration were suppressed by PYSer. Moreover, PYSer showed suppressive activity against the UV-B-induced increase in glycosaminoglycans (GAG). These results indicate that PYSer is a promising antioxidant for the prevention of chronic skin photoaging by its iron-sequestering activity.     

Quantitative and bioluminescent assay to measure efficacy of conventional and DNA vaccinations against Helicobacter pylori

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP-PE in a liposome formulation. To determine the effectiveness of a vaccine against H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum of live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if vaccine candidates are protective. To overcome this problem, we developed two assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a non-competitive direct quantitative PCR using a highly sensitive bioluminescent readout. The competitive PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H. pylori by either the bioluminescent assay or the competitive PCR was reliable, specific and sensitive compared to quantitative growth assays which often gave false results. The bioluminescent assay was much more sensitive and less labor/time intensive than the competitive PCR. The bioluminescent assay was able to quantitate as few as 100 bacteria, while the competitive assay could not detect less than 10(3) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research applications, such as the development of vaccines, pathogenesis of gastric disease and monitoring of antibiotic treatment.     

Advantages and limitations of on-line solid phase extraction coupled to liquid chromatography-mass spectrometry technologies versus biosensors for monitoring of emerging contaminants in water

On-line solid phase extraction (SPE) coupled to liquid chromatography-mass spectrometry (LC-MS) and biosensors are advanced technologies that have found increasing application in the analysis of environmental contaminants although their application to the determination of emerging contaminants (previously unknown or unrecognized pollutants) has been still limited. This review covers the most recent advances occurred in the areas of on-line SPE-LC-MS and biosensors, discusses and compares the main strengths and limitations of the two approaches, and examines their most relevant applications to the analysis of emerging contaminants in environmental waters. So far, the on-line configuration most frequently used has been SPE coupled to liquid chromatography-(tandem) mass spectrometry. Sorbents used for on-line SPE have included both traditional (alkyl-bonded silicas and polymers) and novel (restricted access materials (RAMs), molecularly imprinted synthetic polymers (MIPs), and immobilized receptors or antibodies (immunosorbents) materials. The biosensor technologies most frequently applied have been based on the use of antibodies and, to a lesser extent, enzymes, bacteria, receptors and DNA as recognition elements, and the use of optical and electrochemical transducing elements. Emerging contaminants investigated by means of these two techniques have included pharmaceuticals, endocrine disrupting compounds such as estrogens, alkylphenols and bisphenol A, pesticides transformation products, disinfection by-products, and bacterial toxins and mycotoxins, among others. Both techniques offer advantageous, and frequently comparable, features such as high sensitivity and selectivity, minimum sample manipulation, and automation. Biosensors are, in addition, relatively cheap and fast, which make them ideally suited for routine testing and screening of samples; however, in most cases, they can not compete yet with on-line SPE procedures in terms of accuracy, reproducibility, reliability (confirmation) of results, and capacity for multi-analyte determination.     

Dietary beta-carotene and 13-cis-retinoic acid are not effective in preventing some features of UVB-induced dermal damage in hairless mice

Albino hairless mice were fed diets containing 10 g/kg feed of beta-carotene and 200 mg/kg feed of 13-cis retinoic acid to assess the ability of these molecules to prevent UVB-induced dermal damage. Diets were administered for 12 weeks prior to UVB exposure and were continued throughout the 20 week irradiation period. The UVB source was a bank of FS-20 sunlamps (280-400 nm: peak 313 nm). Exposures were thrice weekly at 0.1 J/cm2 per exposure for the first 10 weeks and 0.2 J/cm2 per exposure for the second 10 weeks. Histologic evaluation of skin biopsies revealed no difference, between animals fed active or placebo diets, in UVB-induced elastosis, collagen changes or amounts of glycosaminoglycans and proteoglycans of the ground substance.     

Fractionated illumination after topical application of 5-aminolevulinic acid on normal skin of hairless mice: the influence of the dark interval

We have previously shown that light fractionation during topical aminolevulinic acid based photodynamic therapy (ALA-PDT) with a dark interval of 2h leads to a significant increase in efficacy in both pre-clinical and clinical PDT. However this fractionated illumination scheme required an extended overall treatment time. Therefore we investigated the relationship between the dark interval and PDT response with the aim of reducing the overall treatment time without reducing the efficacy. Five groups of mice were treated with ALA-PDT using a single light fraction or the two-fold illumination scheme with a dark interval of 30 min, 1, 1.5 and 2h. Protoporphyrin IX fluorescence kinetics were monitored during illumination. Visual skin response was monitored in the first seven days after PDT and assessed as PDT response. The PDT response decreases with decreasing length of the dark interval. Only the dark interval of 2h showed significantly more damage compared to all the other dark intervals investigated (P<0.05 compared to 1.5h and P<0.01 compared to 1h, 30 min and a single illumination). No relationship could be shown between the utilized PpIX fluorescence during the two-fold illumination and the PDT response. The rate of photobleaching was comparable for the first and the second light fraction and not dependent of the length of dark interval used. We conclude that in the skin of the hairless mouse the dark interval cannot be reduced below 2h without a significant reduction in PDT efficacy.     

Ecofriendly synthesis of silver nanoparticles using Kei-apple (Dovyalis caffra) fruit and their efficacy against cancer cells and clinical pathogenic microorganisms

Green fabrication has become a safe approach for producing nanoparticles. Plant-based biogenic synthesis of silver nanoparticles (AgNPs) has emerged as a possible alternative to traditional chemical production. In this paper, we provide a low-cost, green synthesis of AgNPs utilizing using Kei-apple (Dovyalis caffra) fruit extract. Ultraviolet–visible (UV–Vis) spectroscopy, Fourier Transform Infrared (FTIR), Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD), Scanning-Electron Microscope (SEM), and Dynamic Light Scattering (DLS) analyses were used to characterize green produced AgNPs. The formation of AgNPs was shown to have a surface resonance peak of 415 nm in UV–visible spectra, and FTIR spectra verified the participation of biological molecules in Synthesis of AgNPs. The TEM revealed that the biosynthesized AgNPs were mostly spherical in form, with size range of 12–53 nm. XRD diffractogram was used to demonstrate the face cubic centre (fcc) character of AgNPs. Excellent anticancer activity of AgNPs was recorded where more than 80% of Prostate Cancer (PC-3) cell lines was inhibited by 100–150 µg/mL of AgNPs, while 38% only was recorded using AgNO₃ and 55.62% was recorded D. caffra fruit extract at 150 µg/mL. Destructions of PC-3 cell was observed as a result of exposed to AgNPs, followed by D. caffra fruit extract, while minor alterations were recorded as exposed to AgNO₃. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging using AgNPs was three fold using fruit extract at 100 µg/mL indicating good antioxidant activity. Excellent inhibitory activity of AgNPs was recorded against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus fumigatus with inhibition diameter zone 28.22 ± 0.25 mm, 23.21 ± 0.35 mm, 27.25 ± 0.03 mm, 28.40 ± 0.15 mm, 29.23 ± 0.44 mm, and 9.52 ± 0.5 mm, respectively compared with AgNO₃. D. caffra fruits considered a promising and safe source for fabrication of AgNPs with multi-biological functions.     

Alterations of hairless mouse skin exposed to chronic UV irradiation and its prevention by hydrocortisone

Ultraviolet-induced alterations of skin were investigated in a murine animal model. Groups of hairless mice were exposed to UV (black light, lambda max 352 nm; UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) for 20 weeks at a dose of 16.3 J/cm2 five times weekly on weekdays. At the end of 20 weeks irradiation, the dorsal skins were biochemically and histologically examined. Ultraviolet caused remarkable increases in amounts of hyaluronan, chondroitin sulfates and dermatan sulfates in skin (microgram/cm2). Interestingly, a significant change in a collagen content (hydroxyproline, microgram/g of dry powder) caused by UV irradiation was not observed, whereas the amount of collagen (hydroxyproline, microgram/cm2) increased remarkably. Histologically, no distinguishable thickening was observed in both upper dermis and lower dermis, but thickening of the epidermis was observed. Furthermore, the histological study indicated that UV irradiation caused a disappearance of crowds of adipocytes, alternative appearance of numerous fibroblasts and accumulation of collagen bundles and hyaluronan in lower dermis. Hydrocortisone, an anti-inflammatory agent, prevented both the fibrosis of lower dermis and the accumulation of the extracellular matrix components. Based on these results, it seems reasonable that UV penetrates into the lower dermis and causes fibrosis there, resulting from the inflammatory responses.     

Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg(-1) day(-1) to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2-5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).