Long-term exposure to commercially available sunscreens containing nanoparticles of TiO2 and ZnO revealed no biological impact in a hairless mouse model.

BACKGROUND: The application of sunscreen is a critical component of a sun-safe strategy, however the possibility of unexpected, adverse outcomes resulting from long-term use of sunscreens containing nanoparticles of titanium dioxide (TiO2) and zinc oxide (ZnO) has not yet been examined. Here, immune-competent hairless mice were exposed over a 36-week period to weekly topical applications of sunscreens containing nanoparticles of ZnO or TiO2, or no metal oxide nanoparticles, with or without subsequent exposure to ultraviolet radiation (UVR). Control groups received no sunscreen applications, with or without UVR. RESULTS: Mice exposed to UVR in the absence of sunscreen developed statistically significant incidences of histologically-diagnosed malignant and benign skin neoplasms, whereas no statistically significant adverse biological outcomes were found in mice treated with the sunscreens containing ZnO or TiO2 nanoparticles. Elevated levels of Ti were detected in the livers of mice treated with sunscreen containing TiO2 nanoparticles compared to untreated control, but total Zn concentrations did not significantly alter in any major organs except for the skin of mice treated with ZnO sunscreen. Exposure to UVR did not have a significant impact on examined tissue concentrations of Zn or Ti. Few to no transcriptional changes were found in ZnO or TiO2-treated groups, but mice treated with the sunscreen containing only organic filters showed substantial gene disregulation. CONCLUSIONS: Taken together with previous work, this long-term study provided no basis to avoid the use of sunscreens containing metal oxide nanoparticles.

Size-dependent cytotoxicity and genotoxicity of ZnO particles to human lymphoblastoid (WIL2-NS) cells.

The relationship between particle size and cytogenotoxicity of ZnO particles was systematically studied in vitro using WIL2-NS human lymphoblastoid cells. Before toxicity measurements, the ZnO particles of three different sizes (26 nm, 78 nm, and 147 nm) were well characterized for their physical and chemical properties to ensure that variations in other properties including surface chemistry and particle shape, which also may influence particle toxicity, were minimal. Cell viability testing showed that increasing cytotoxicity was associated with decreasing particle size. Both the dissolution kinetics of ZnO particles in supplemented cell culture medium and the apparent numbers of ZnO particles internalized by cells were size dependent and showed strong correlation with cytotoxicity. Genotoxicity, as measured by micronucleus formation, was significantly enhanced in the presence of the medium-sized and large-sized particles. The observation that necrosis increased with smaller- sized particles but micronuclei were present to a greater extent with larger- sized particles suggests that different mechanisms of cell damage induction or susceptibilities are operating depending on particle size.

A review of critical factors for assessing the dermal absorption of metal oxide nanoparticles from sunscreens applied to humans, and a research strategy to address current deficiencies.

Metal oxide nanoparticles in sunscreens provide broad-spectrum ultraviolet protection to skin. All studies to assess dermal penetration of nanoparticles have unanimously concluded that the overwhelming majority of nanoparticles remain on the outer surface of the skin. However, possibly due to many different experimental protocols in use, conclusions over the potential penetration to viable skin are mixed. Here, we review several factors that may influence experimental results for dermal penetration including the species studied (human, or animal model), size and coating of the metal oxide nanoparticles, composition of the sunscreen formulation, site of sunscreen application, dose and number of applications, duration of the study, types of biological samples analysed, methods for analysing samples, exposure to UV and skin flexing. Based on this information, we suggest an appropriate research agenda involving international collaboration that maximises the potential for dermal absorption of nanoparticles, and their detection, under normal conditions of sunscreen use by humans. If results from this research agenda indicate no absorption is observed, then concerns over adverse health effects from the dermal absorption of nanoparticles in sunscreens may be allayed.

Detecting free radicals in sunscreens exposed to UVA radiation using chemiluminescence.

One of the current concerns with the application of nanoparticles in sunscreens, and in particular nano-TiO2 and ZnO, is their potential to photogenerate free radicals and reactive oxygen species (ROS) when they absorb ultraviolet wavelengths from sunlight. Free radicals and ROS are known to be associated with UV-induced skin damage and oxidative stress, from which sunscreens are expected to offer significant protection. Here we describe a simple method, based on chemiluminescence emission, for detecting free radicals generated in commercial sunscreens alone, and when applied to various substrates, following exposure to UVA (320-400nm) radiation. This photo-induced chemiluminescence (PICL) technique could be used to optimise sunscreen formulations so as to minimise free radical photogeneration during exposure to sunlight.

Dermal absorption and short-term biological impact in hairless mice from sunscreens containing zinc oxide nano- or larger particles.

Previous studies have shown no, or very limited, skin penetration of metal oxide nanoparticles following topical application of sunscreens, yet concerns remain about their safety compared to larger particles. Here, we assessed the comparative dermal absorption of a traceable form of Zn ((68)Zn) from (68)ZnO nano-sized and larger particles in sunscreens. Sunscreens were applied to the backs of virgin or pregnant hairless mice over four days. Control groups received topical applications of the sunscreen formulation containing no ZnO particles, or no treatment. Major organs were assessed for changes in (68)Zn/(64)Zn ratios, (68)Zn tracer and total Zn concentrations. Short-term biological impact was assessed by measuring levels of serum amyloid A in blood, and by performing whole-genome transcriptional profiling on livers from each group. Increased concentrations of (68)Zn tracer were detected in internal organs of mice receiving topical applications of (68)ZnO (nano-sized and larger particles), as well as in fetal livers from treated dams, compared with controls. Furthermore, concentrations of (68)Zn in organs of virgin mice treated with sunscreen containing (68)ZnO nanoparticles were found to be significantly higher than in mice treated with sunscreen containing larger (68)ZnO particles. However, no ZnO-mediated change in total Zn concentration in any of the major organs was observed. Thus, despite (68)Zn absorption, which may have been in the form of soluble (68)Zn species or (68)ZnO particles (not known), Zn homeostasis was largely maintained, and the presence of ZnO particles in sunscreen did not elicit an adverse biological response in the mice following short-term topical applications.

Surface coatings of ZnO nanoparticles mitigate differentially a host of transcriptional, protein and signalling responses in primary human olfactory cells.

BACKGROUND: Inhaled nanoparticles have been reported in some instances to translocate from the nostril to the olfactory bulb in exposed rats. In close proximity to the olfactory bulb is the olfactory mucosa, within which resides a niche of multipotent cells. Cells isolated from this area may provide a relevant in vitro system to investigate potential effects of workplace exposure to inhaled zinc oxide nanoparticles. METHODS: Four types of commercially-available zinc oxide (ZnO) nanoparticles, two coated and two uncoated, were examined for their effects on primary human cells cultured from the olfactory mucosa. Human olfactory neurosphere-derived (hONS) cells from healthy adult donors were analyzed for modulation of cytokine levels, activation of intracellular signalling pathways, changes in gene-expression patterns across the whole genome, and compromised cellular function over a 24 h period following exposure to the nanoparticles suspended in cell culture medium. RESULTS: ZnO nanoparticle toxicity in hONS cells was mediated through a battery of mechanisms largely related to cell stress, inflammatory response and apoptosis, but not activation of mechanisms that repair damaged DNA. Surface coatings on the ZnO nanoparticles mitigated these cellular responses to varying degrees. CONCLUSIONS: The results indicate that care should be taken in the workplace to minimize generation of, and exposure to, aerosols of uncoated ZnO nanoparticles, given the adverse responses reported here using multipotent cells derived from the olfactory mucosa.

Applying quantitative structure-activity relationship approaches to nanotoxicology: current status and future potential.

The potential (eco)toxicological hazard posed by engineered nanoparticles is a major scientific and societal concern since several industrial sectors (e.g. electronics, biomedicine, and cosmetics) are exploiting the innovative properties of nanostructures resulting in their large-scale production. Many consumer products contain nanomaterials and, given their complex life-cycle, it is essential to anticipate their (eco)toxicological properties in a fast and inexpensive way in order to mitigate adverse effects on human health and the environment. In this context, the application of the structure-toxicity paradigm to nanomaterials represents a promising approach. Indeed, according to this paradigm, it is possible to predict toxicological effects induced by chemicals on the basis of their structural similarity with chemicals for which toxicological endpoints have been previously measured. These structure-toxicity relationships can be quantitative or qualitative in nature and they can predict toxicological effects directly from the physicochemical properties of the entities (e.g. nanoparticles) of interest. Therefore, this approach can aid in prioritizing resources in toxicological investigations while reducing the ethical and monetary costs that are related to animal testing. The purpose of this review is to provide a summary of recent key advances in the field of QSAR modelling of nanomaterial toxicity, to identify the major gaps in research required to accelerate the use of quantitative structure-activity relationship (QSAR) methods, and to provide a roadmap for future research needed to achieve QSAR models useful for regulatory purposes.

Azide photochemistry for facile modification of graphitic surfaces: preparation of DNA-coated carbon nanotubes for biosensing.

A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms-as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions.

Comparison of dermal absorption of zinc from different sunscreen formulations and differing UV exposure based on stable isotope tracing.

In a pilot study to determine if zinc (Zn) from zinc oxide nanoparticles in sunscreen can penetrate human skin in vivo, nanoparticles (~30nm) of a stable isotope (52% (68)Zn enrichment) were incorporated into an essentially phytochemical-based formulation and applied to the backs of 3 human subjects twice daily for 5 days during the Southern Hemisphere winter. Blood and urine were collected prior to application and at regular intervals and up to 50 days. As observed in a larger outdoor trial following this pilot study but with a different formulation and with UV exposure: values of (68)Zn in blood continued to increase beyond the 5 day application phase with the highest measurement at 14 days after the first application; variable amounts of the (68)Zn tracer were observed in urine; and the amounts of extra Zn added to blood were small and indicate very low levels of absorption (minimal estimate <0.01% of the applied dose) through the skin. Reasons for differences in absorption detected in the stable isotope trials and previous investigations include: the sensitivity of the stable isotope method; the duration of the investigations; the number of applications of sunscreen formulation; in vitro methods with excised skin; lack of measurement of blood and urine; no skin flexing; and lack of UV exposure.

Durability and inflammogenic impact of carbon nanotubes compared with asbestos fibres.

BACKGROUND: It has been suggested that carbon nanotubes might conform to the fibre pathogenicity paradigm that explains the toxicities of asbestos and other fibres on a continuum based on length, aspect ratio and biopersistence. Some types of carbon nanotubes satisfy the first two aspects of the fibre paradigm but only recently has their biopersistence begun to be investigated. Biopersistence is complex and requires in vivo testing and analysis. However durability, the chemical mimicking of the process of fibre dissolution using in vitro treatment, is closely related to biopersistence and more readily determined. Here, we describe an experimental process to determine the durability of four types of carbon nanotubes in simulated biological fluid (Gambles solution), and their subsequent pathogenicity in vivo using a mouse model sensitive to inflammogenic effects of fibres. The in vitro and in vivo results were compared with well-characterised glass wool and asbestos fibre controls. RESULTS: After incubation for up to 24 weeks in Gambles solution, our control fibres were recovered at percentages consistent with their known in vitro durabilities and/or in vivo persistence, and three out of the four types of carbon nanotubes tested (single-walled (CNTSW) and multi-walled (CNTTANG2, CNTSPIN)) showed no, or minimal, loss of mass or change in fibre length or morphology when examined by electron microscopy. However, the fourth type [multi-walled (CNTLONG1)] lost 30% of its original mass within the first three weeks of incubation, after which there was no further loss. Electron microscopy of CNTLONG1 samples incubated for 10 weeks confirmed that the proportion of long fibres had decreased compared to samples briefly exposed to the Gambles solution. This loss of mass and fibre shortening was accompanied by a loss of pathogenicity when injected into the peritoneal cavities of C57Bl/6 mice compared to fibres incubated briefly. CNTSW did not elicit an inflammogenic effect in the peritoneal cavity assay used here. CONCLUSIONS: These results support the view that carbon nanotubes are generally durable but may be subject to bio-modification in a sample-specific manner. They also suggest that pristine carbon nanotubes, either individually or in rope-like aggregates of sufficient length and aspect ratio, can induce asbestos-like responses in mice, but that the effect may be mitigated for certain types that are less durable in biological systems. Results indicate that durable carbon nanotubes that are either short or form tightly bundled aggregates with no isolated long fibres are less inflammogenic in fibre-specific assays.

Surface modifications of ZnO nanoparticles and their cytotoxicity.

ZnO is a well-known UV absorber. At small particle sizes its absorption efficiency is substantially increased and this property, combined with transparency to visible light, has attracted growing interest in its applications in personal care products such as sunscreens. However, some recent studies suggest that ZnO nanoparticles could induce considerable toxicity to certain cells and microorganisms. Aiming to reduce cytotoxicity of ZnO nanoparticles without impairing their unique properties, this paper examines the influence of surface modifications to ZnO nanoparticles using coatings such as silica (SiO2) and Poly methyl Acrylic Acid (PMAA). It was found that both PMAA and SiO2 coatings were physically attached to the ZnO surface and their presence did not weaken UV absorption of the original nanoparticles. Uncoated ZnO and SiO2-coated ZnO exhibited similar cytotoxicity to human lymphoblastoid cells, while PMAA-coated ZnO nanoparticles had little adverse effect except at high concentrations. The type of coating was also shown to affect the generation of Reactive Oxygen Species (ROS). The correlation between cell viability and ROS level led to conclusions that enhanced oxidative stress could be one of the reasons for cytotoxicity of ZnO nanoparticles.

Effects of surface chemistry on cytotoxicity, genotoxicity, and the generation of reactive oxygen species induced by ZnO nanoparticles.

The relationship between the toxicity of zinc oxide (ZnO) nanoparticles (NPs) and their surface chemistry was investigated. Cytotoxicity, genotoxicity, and the ability to generate reactive oxygen species (ROS) were assessed for well-characterized ZnO NPs whose surface chemistry was varied from its pristine state by coating with oleic acid (OA), poly(methacrylic acid) (PMAA), or components adsorbed from cell culture medium (medium-soaked). It was found that uncoated NPs showed ROS accumulation and diminished cell viability whereas all tested surface coatings assisted in reducing ROS production and cytotoxicity. The ability of coatings to reduce the cytotoxicity of ZnO NPs was ranked in the following order: medium-soaked ≈ PMAA > OA. However, PMAA-coated ZnO had significant genotoxicity compared to uncoated ZnO and the other coated NPs, highlighting the need to investigate thoroughly the effects of NP surface modification on both cytotoxicity and genotoxicity assays. The lowest toxicity was achieved with a surface coating of components from a cell culture medium.

Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard.

Sunscreens containing metal oxide nanoparticles appear transparent on the skin and provide excellent protection against sunburn caused by UV radiation. While it is likely that nanoparticles remain on the surface of the skin of healthy adult humans, and thus are considered safe for use in sunscreens, there has been no comprehensive assessment of the impact on human health from exposure to the metal oxide nanoparticles destined for use in sunscreens, either in the workplace during the manufacturing process, in long-term use across a range of skin conditions, or upon release into the broader environment, either accidentally or consequent of normal sunscreen use. In this review, we focus on zinc oxide nanoparticles destined for use in modern sunscreens, and discuss the potential for human exposure and the health hazard at each stage of their manufacture and use. We highlight where there is a need for further research.

Small amounts of zinc from zinc oxide particles in sunscreens applied outdoors are absorbed through human skin.

Metal oxide nanoparticles are commonly used in personal-care formulations as protective agents against exposure to ultraviolet radiation. Although previous research has concluded that nanoparticles do not penetrate healthy skin, it remains contentious whether this conclusion holds under normal conditions of sunscreen use. Humans (n = 20) were exposed to sunscreens containing zinc oxide (ZnO) particles to determine if Zn from the particles was absorbed through skin over five consecutive days under outdoor conditions. Two sunscreens were tested-"nano sunscreen" containing 19-nm nanoparticles and "bulk sunscreen" containing > 100-nm particles. Venous blood and urine samples were collected 8 days before exposure, twice daily during the trial, and 6 days post-exposure. As the first application in nanotechnology studies, stable isotope tracing was used where the ZnO, enriched to > 99% with the stable isotope (68)Zn, allowed dermally absorbed zinc to be distinguished from naturally occurring zinc. The overwhelming majority of applied (68)Zn was not absorbed, although blood and urine samples from all subjects exhibited small increases in levels of tracer (68)Zn. The amount of tracer detected in blood after the 5-day application period was ∼1/1000 th that of total Zn in the blood compartment. Tracer levels in blood continued to increase beyond the 5-day application phase in contrast to those in urine. Levels of (68)Zn in blood and urine from females receiving the nano sunscreen appeared to be higher than males receiving the same treatment and higher than all subjects receiving the bulk sunscreen. It is not known whether (68)Zn has been absorbed as ZnO particles or soluble Zn or both.

Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability.

BACKGROUND: Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues. RESULTS: A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent. CONCLUSION: Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum.