Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity

The lack of correlative and predictive models to assess acute and chronic toxicities limits the rapid pre-clinical development of new therapeutics. This barrier is due in part to the exponential growth of nanotechnology and nanotherapeutics, coupled with the lack of rigorous and robust screening assays and putative standards. It is a fairly simple and cost-effective process to initially screen the toxicity of a nanomaterial by using invitro cell cultures; unfortunately it is nearly impossible to imitate a complimentary invivo system. Small mammalian models are the most common method used to assess possible toxicities and biodistribution of nanomaterials in humans. Alternatively, Daniorerio, commonly known as zebrafish, are proving to be a quick, cheap, and facile model to conservatively assess toxicity of nanomaterials.     

Editorial

The square-wave voltammetry of isolated isomers of C₈₄ is reported. Based on their relative abundance, the isomers are assumed to have D₂ and D_2d symmetry. The D₂ isomer has four reversible reductions at ?0.65, ?0.98, ?1.34, and ?1.75 V versus Fc/Fc⁺ prior to electrolyte decomposition. This behavior is consistent with previous reports, and is similar to the voltammetry of C₆₀ and C₇₀ (e.g. sequential reductions spaced at slightly increasing intervals). The D_2d isomer has five reversible reductions at ?0.46, ?0.77, ?1.58, ?1.98, and ?2.27 V versus Fc/Fc⁺ prior to the onset of electrolyte decomposition. The large potential difference separating the second and third reductions of the D_2d isomer is an aspect of the voltammetry not reported when the D_2d isomer was analyzed as part of a mixture of C₈₄ isomers (P.L. Boulas, M.T. Jones, R.S. Ruoff et al., J. Phys. Chem. 100 (1996) 7573–7579). The large potential difference between the second and third reductions of the D_2d isomer of C₈₄ is similar to the voltammetric behavior of C₈₂.     

Skin penetration and kinetics of pristine fullerenes (C60) topically exposed in industrial organic solvents

Pristine fullerenes (C₆₀) in different solvents will be used in many industrial and pharmaceutical manufacturing and derivatizing processes. This report explores the impact of solvents on skin penetration of C₆₀ from different types of industrial solvents (toluene, cyclohexane, chloroform and mineral oil). Yorkshire weanling pigs (n = 3) were topically dosed with 500 μL of 200 μg/mL C₆₀ in a given solvent for 24 h and re-dosed daily for 4 days to simulate the worst scenario in occupational exposures. The dose sites were tape-stripped and skin biopsies were taken after 26 tape-strips for quantitative analysis. When dosed in toluene, cyclohexane or chloroform, pristine fullerenes penetrated deeply into the stratum corneum, the primary barrier of skin. More C₆₀ was detected in the stratum corneum when dosed in chloroform compared to toluene or cyclohexane. Fullerenes were not detected in the skin when dosed in mineral oil. This is the first direct evidence of solvent effects on the skin penetration of pristine fullerenes. The penetration of C₆₀ into the stratum corneum was verified using isolated stratum corneum in vitro; the solvent effects on the stratum corneum absorption of C₆₀ were consistent with those observed in vivo. In vitro flow-through diffusion cell experiments were conducted in pig skin and fullerenes were not detected in the receptor solutions by 24 h. The limit of detection was 0.001 μg/mL of fullerenes in 2 mL of the receptor solutions.     

Development of water-soluble metallofullerenes as X-ray contrast media

Fullerenes are a new carbonic allotrope having a cage structure. We investigated whether fullerenes containing one or two atoms of heavy metals could be an X-ray contrast material with little adverse effects. One or two atoms of dysprosium (Dy), erbium (Er), gadolinium (Gd), europium (Eu) and lutetium (Lu) were encapsulated into fullerene (C₈₂), which was synthesized as a polyhydroxyl form (e.g., Gd@C₈₂(OH)n, n=40, Gd – fullerenols). They were dissolved in water at maximum soluble concentrations and subjected to CT number analysis. The CT numbers of the solutions were measured using a 4- or 16-row multidetector CT scanner. The CT number of the water-soluble metallofullerenes were 56.0 HU for Dy@C₈₂(OH)₄₀, 111.5 HU for Er@C₈₂(OH)₄₀, 58.4 HU for Gd@C₈₂(OH)₄₀, 100.9 HU for Eu@C₈₂(OH)₄₀ and 23.3 HU for Lu₂@C₈₂(OH)₄₀. The CT numbers of the metallofullerenes investigated in the present study were not high enough to be used in the clinic in place of iodinated contrast materials. However, if nanotechnology progresses in the near future, it may prove to have a possibility as an X-ray contrast material.     

Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C₆₀(OH)_22-26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C₆₀(OH)_22-26 in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 μM. Exposure to an 8.5 J·cm^− 2 dose of visible light in the presence of > 5 μM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 μM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of Φ = 0.05 in D₂O and with a range of 0.002-0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage.     

Review of fullerene toxicity and exposure – Appraisal of a human health risk assessment, based on open literature

Fullerenes have gained considerable attention due to their anti-oxidant and radical scavenging properties. Their current applications include targeted drug delivery, energy application, polymer modifications and cosmetic products. The production of fullerenes and their use in consumer products is expected to increase in future.     

Environmental behavior and ecotoxicity of engineered nanoparticles to algae,plants, and fungi

Developments in nanotechnology are leading to a rapid proliferation of new materials that are likely to become a source of engineered nanoparticles (ENPs) to the environment, where their possible ecotoxicological impacts remain unknown. The surface properties of ENPs are of essential importance for their aggregation behavior, and thus for their mobility in aquatic and terrestrial systems and for their interactions with algae, plants and, fungi. Interactions of ENPs with natural organic matter have to be considered as well, as those will alter the ENPs aggregation behavior in surface waters or in soils. Cells of plants, algae, and fungi possess cell walls that constitute a primary site for interaction and a barrier for the entrance of ENPs. Mechanisms allowing ENPs to pass through cell walls and membranes are as yet poorly understood. Inside cells, ENPs might directly provoke alterations of membranes and other cell structures and molecules, as well as protective mechanisms. Indirect effects of ENPs depend on their chemical and physical properties and may include physical restraints (clogging effects), solubilization of toxic ENP compounds, or production of reactive oxygen species. Many questions regarding the bioavailability of ENPs, their uptake by algae, plants, and fungi and the toxicity mechanisms remain to be elucidated.     

Determination of heterogeneous rate constants for the electrochemical reduction of fullerene C60 by the method of cyclic voltammetry on a hemispherical ultramicroelectrode

A sealed cell for the study of low temperature electrochemistry is described. From room temperature to 223 K the kinetic properties of the reduction of fullerene C₆₀ in CH₃CN + toluene (volume ratio 1:5) containing 0.1 M TBAPF₆ were studied. Six one-electron reduction waves of C₆₀ have been obtained under our experimental conditions. The results show that the diffusion activation energy of C₆₀ is 18.0 kJ mol^?1. The heterogeneous rate constants of the first five redox couples of C₆₀ have been measured at different temperatures. The activation energies for the first five electroreductions were evaluated.     

Fullerene antioxidants decrease organophosphate-induced acetylcholinesterase inhibition in vitro

Although organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition is the critical mechanism causing toxicities that follow exposure, other biochemical events, including oxidative stress, have been reported to contribute to OP toxicity. Fullerenes are carbon spheres with antioxidant activity. Thus, we hypothesized that fullerenes could counteract the effects of OP compounds and tested this hypothesis using two in vitro test systems, hen brain and human neuroblastoma SH-SY5Y cells. Cells were incubated with eight different derivatized fullerene compounds before challenge with paraoxon (0 = control, 5 × 10⁻⁸, 10⁻⁷, 2 × 10⁻⁷ or 5 × 10⁻⁷ M) or diisopropylphosphorofluoridate (DFP, 0 = control, 5 × 10⁻⁶, 10⁻⁵, 2 × 10⁻⁵, and 5 × 10⁻⁵ M) and measurement of AChE activities. Activities of brain and SH-SY5Y AChE with OP compounds alone ranged from 55-83% lower than non-treated controls after paraoxon and from 60-92% lower than non-treated controls after DFP. Most incubations containing 1 and 10 μM fullerene derivatives brought AChE activity closer to untreated controls, with improvements in AChE activity often >20%. Using dissipation of superoxide anion radicals as an indicator (xanthine oxidation as a positive control), all fullerene derivatives demonstrated significant antioxidant capability in neuroblastoma cells at 1 μM concentrations. No fullerene derivative at 1 μM significantly affected neuroblastoma cell viability, when determined using either Alamar Blue dye retention or a luminescent assay for ATP production. These studies suggest that derivatized fullerene nanomaterials have potential capability to ameliorate OP-induced AChE inhibition resulting in toxicities.     

Time-dependent variation in the biodistribution of C₆₀ in rats determined by liquid chromatography-tandem mass spectrometry

We examined the biodistribution of C₆₀ in rats after tail vein administration using LC-MS/MS. C₆₀ was detected in various tissues, such as brain, kidneys, liver, lungs, and spleen of rats. On the other hand, no C₆₀ was found in blood. The highest C₆₀ concentration was observed in the lungs, followed by spleen, liver, kidneys, and brain. These results suggested that C₆₀ injected in the tail vein could be filtered by lung capillary vessels and accumulate in the lungs prior to being distributed to other tissues. Moreover, C₆₀ not being detected in the blood indicates that clearance of C₆₀ from the blood by filtration might effectively occur in the lungs. The time-dependent variation in the biodistribution of C₆₀ was evaluated. A time-dependent decrease in C₆₀ concentrations was observed in all tissues, except spleen. Moreover, a decreasing trend of C₆₀ levels differed among tissues, which could be due to differences in accumulation. These results suggest that unmodified C₆₀ and/or C₆₀ metabolites by metabolic enzymes could be excreted into feces and/or urine. In further studies, the metabolic and excretion pathways of C₆₀ should be evaluated to understand the toxicokinetics of C₆₀.