Assessment of the in vitro dermal irritation potential of cerium,silver, and titanium nanoparticles in a human skin equivalent model

Metal nanoparticles can potentially contact human skin during their manufacture and use in commercial products. This study examined the potential of metal nanoparticles to elicit irritant contact dermatitis in a human skin equivalent model (HSEM) derived from keratinocytes. Ag (10–100?nm), TiO₂ (22–214?nm), and CeO₂ (15–40?nm) nanoparticles were studied. The Ag particles were either coated/shelled with silica or capped with citrate or polyvinylpyrrolidone and were in water. The TiO₂ and CeO₂ particles were suspended in media containing 10% fetal bovine serum. The particles (1?mg/ml) were applied to the epidermal surface of the HSEM. Positive (5% sodium dodecyl sulfate (SDS)) and negative controls (saline or media) were included. After 1-h exposure at 37?°C, the HSEM was washed with saline to remove the nanoparticles. Following a 42-h incubation (37?°C), HSEM viability was assessed using the MTT assay. A test substance is considered a dermal irritant if the HSEM viability is?2, and CeO₂ nanoparticles examined were not dermal irritants under the conditions used in this study. The stratum corneum of the HSEM may limit penetration of metal nanoparticles to induce toxicity.     

In vitro and in vivo comparison of dermal irritancy of jet fuel exposure using EpiDerm (EPI-200) cultured human skin and hairless rats

The purpose of this study was to evaluate an in vitro EpiDerm human skin model (EPI-200) to study the irritation potential of jet fuels (JP-8 and JP-8+100). Parallel in vivo studies on hairless rats on the dermal irritancy of jet fuels were also conducted. Cytokines are an important part of an irritation and inflammatory cascade, which are expressed in upon dermal exposures of irritant chemicals even when there are no obvious visible marks of irritation on the skin. We have chosen two primary cytokines (IL-1alpha and TNF-1alpha) as markers of irritation response of jet fuels. Initially, the EPI-200 was treated with different quantities of JP-8 and JP-8+100 to determine quantities which did not cause significant cytotoxicity, as monitored using the MTT assay and paraffin embedded histological cross-sections. Volumes of 2.5-50 microl/tissue (approximately 4.0-78 microl/cm2) of JP-8 and JP-8+100 showed a dose dependent loss of tissue viability and morphological alterations of the tissue. At a quantity of 1.25 microl/tissue (approximately 2.0 microl/cm2), no significant change in tissue viability or morphology was observed for exposure time extending to 48 h. Nonetheless, this dose induced significant increase in IL-1alpha and TNF-alpha release versus non-treated controls after 24 and 48 h. In addition, IL-1alpha release for JP-8+100 was significantly higher than that observed for JP-8, but TNF-alpha release after 48 h exposure to these two jet fuels was the same. These findings parallel in vivo studies on hairless rats, which indicated higher irritation levels due to JP-8+100 versus JP-8. In vivo, transepidermal water loss (TEWL) and IL-1alpha expression levels followed the order JP-8+100 > JP-8 > control. Further, in vivo TNF-alpha levels for JP-8 and JP-8+100 were also elevated but not significantly different from one another. In aggregate, these findings indicate that EPI-200 tissue model can be utilized as an alternative to the use of animals in evaluating dermal irritation.     

Evaluation of paromomycin sulphate permeation using ex vivo human skin model

Ex vivo evaluation of drug release and skin permeation from topical formulations of antileishmanial drug paromomycin sulphate was carried out using intact full thickness human skin. Potency-based microbiological assay was used for the analysis of paromomycin concentrations. A total percentage drug recovery of 86?±?26% was obtained. Incubation periods of 1 and 3?h resulted in percentage drug permeation into deep skin layers ranging from 1.3?±?0.04% to 5.3?±?2.0% with paraffin-based ointment and from 1.6?±?0.8% to 3.9?±?1% with microemulsion-based emulgel. Although a small percentage, this is still significantly higher than those previously reported using animal skin models.     

An evaluation of thiram toxicity on cultured human skin fibroblasts

Thiram is widely used in agriculture as a fungicide and, to a lesser extent, as a vulcanizing agent in the rubber industry. In spite of the extensive use of thiram, knowledge on its toxicity and health risk remains limited, and few investigations have been performed to assess specific damage at the cellular and subcellular level. We report here the cytotoxic effects of thiram on cultured human skin fibroblasts. Our results demonstrated that thiram exposure induced a dose- and time-dependent decrease in the viable cell recovery with 100% cell death observed with a concentration of 5.0 mg/l. As judged by morphological changes and biochemical criteria, thiram-mediated cell death was not of the apoptotic but seemed to be of the necrotic type. This cell death was not associated with a modification of gene expression of different constituents of the extracellular matrix. A late increase of lactate production was evident after thiram treatment, suggesting a mitochondrial metabolic pathway dysfunction as reported by other authors using similar compounds. However, this phenomenon appeared as a secondary response to the toxic action of thiram. The cytotoxic effect of thiram is possibly due to an oxidant effect inherent to the structure of thiram and the interaction between thiram and vital cellular molecules.     

TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.     

Statistical evaluation of an acute dermal toxicity test using the dermal fixed dose procedure

The conventional method for the assessment of acute dermal toxicity (OECD Test Guideline 402, 1987) uses death of animals as an endpoint to identify the median lethal dose (LD50). A new OECD Testing Guideline called the dermal fixed dose procedure (dermal FDP) is being prepared to provide an alternative to Test Guideline 402. In contrast to Test Guideline 402, the dermal FDP does not provide a point estimate of the LD50, but aims to identify that dose of the substance under investigation that causes clear signs of nonlethal toxicity. This is then used to assign classification according to the new Globally Harmonised System of Classification and Labelling scheme (GHS). The dermal FDP has been validated using statistical modelling rather than by in vivo testing. The statistical modelling approach enables calculation of the probability of each GHS classification and the expected numbers of deaths and animals used in the test for imaginary substances with a range of LD50 values and dose-response curve slopes. This paper describes the dermal FDP and reports the results from the statistical evaluation. It is shown that the procedure will be completed with considerably less death and suffering than guideline 402, and will classify substances either in the same or a more stringent GHS class than that assigned on the basis of the LD50 value.     

Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo

This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.     

Effect of squalene monohydroperoxide on cytotoxicity and cytokine release in a three-dimensional human skin model and human epidermal keratinocytes

In order to clarify that squalene monohydroperoxide (SQOOH) correlates with changes in morphology through cytotoxicity and establish in vitro evaluation of the cytotoxicity of lipid hydroperoxide, the effect of SQOOH on cytotoxicity and morphology in normal human epidermal keratinocytes (NHEK(B)) and the Gunze three-dimensional cultured human skin model (Vitrolife-skin) was investigated. Additionally, the effect of radical scavengers (mannitol, vitamin C and (+)-catechin) on the cytotoxicity in Vitrolife-skin was studied. The level of lipid hydroperoxide (phoshatidylcholine hydroperoxide: PCOOH) in the cellular membrane was increased with the concentration of SQOOH, and the rise in cytotoxicitv in NHEK(B) was associated with changes to the cellular membrane. A concentration-dependent and protective effect on the increase in cytotoxicity and PCOOH content was observed. To clarify the effect of SQOOH on the release of cytokine from cells, IL-2 level from NHEK(B) and Vitrolife-skin were investigated. IL-2 release from the cells was enhanced by SQOOH and increased at a non-cytotoxic dose. These results suggest that the increase in lipid hydroperoxides resulting from the auto-oxidation of lipids within cellular membranes in the presence of SQOOH correlates with changes in morphology due to cytotoxicity. SQOOH enhanced the release from cells at a non-cytotoxic dose. A method for assessing the protective effect on the cytotoxicity of lipid hydroperoxides using cells would be useful for in vitro evaluation of the cytotoxicity.     

Assessment of dermal safety of oil extracted from Periplaneta americana: acute dermal toxicity,irritation, and sensitization

Abstract

Introduction

The American cockroach (Periplaneta americana) is used in traditional Chinese medicine. Periplaneta americana (P. americana) is rich in oil that has shown potential antioxidant and antibacterial activities in vitro.     

Grafting of human autogenous keratinocytes cultured in vitro

A rapid method of producing epithelial material suitable for covering skin defects after burns and avulsions is presented. In vitro grown keratinocytes suspended as single cells in human plasma were applied to the wound surface and covered with distended meshed xenograft. In this way it was possible in five clinical cases to obtain 25-80% epithelial coverage of wounds up to 200 times as wide as the donor area.     

Genotoxicity,acute oral and dermal toxicity,eye and dermal irritation and corrosion and skin sensitisation evaluation of silver nanoparticles

Abstract

To clarify the health risks related to silver nanoparticles (Ag-NPs), we evaluated the genotoxicity, acute oral and dermal toxicity, eye irritation, dermal irritation and corrosion and skin sensitisation of commercially manufactured Ag-NPs according to the OECD test guidelines and GLP. The Ag-NPs were not found to induce genotoxicity in a bacterial reverse mutation test and chromosomal aberration test, although some cytotoxicity was observed. In acute oral and dermal toxicity tests using rats, none of the rats showed any abnormal signs or mortality at a dose level of ～ 2000 mg/kg. Similarly, acute eye and dermal irritation and corrosion tests using rabbits revealed no significant clinical signs or mortality and no acute irritation or corrosion reaction for the eyes and skin. In a skin sensitisation test using guinea pigs, one animal (1/20) showed discrete or patchy erythema, thus Ag-NPs can be classified as a weak skin sensitiser.     

Effects of selected arginine analogues on sulphur mustard toxicity in human and hairless guinea pig skin keratinocytes

The toxicity of sulphur mustard (HD) was characterized in first passage cultures of human neonatal foreskin keratinocytes and then several arginine analogues were investigated to ascertain their efficacies in protecting against HD toxicity in this system. d- and l-nitroarginine methyl ester (d/l-NAME), l-phosphoarginine, and l-nitroarginine were all found to confer concentration-related protective effects against HD in confluent cultures. l-NAME was used to further characterize this protection and was only effective in cultures that were not actively proliferating. This compound was also found to be efficacious when added to the cultures up to several hours after HD exposure, although its continued presence was required in order for protection to be effective. The protective effects of l-thiocitrulline (l-TC) against HD toxicity were also assessed. This arginine analogue was extremely potent in preventing HD toxicity in actively proliferating, just-confluent, and postconfluent cultures in a concentration-dependent fashion (0.1-15 mM), with little HD toxicity apparent at high l-TC concentrations. Protection was prophylactic in nature, with l-TC having almost maximal effect when added to the cultures only 1 min prior to HD culture exposure. Efficacy then declined rapidly so that no protection was evident when l-TC was added 30 min post-HD. The effects of this drug were persistent, with no decrease in protective efficacy up to 4 days after HD exposure, even when l-TC was removed from the cultures. l-TC did not protect against the antimitotic effects of HD; while l-TC-protected cells were subcultured successfully, they displayed no clonogenic activity. Although l-TC is closely related structurally to protective nitroarginine derivatives, the characteristics of l-TC protection against HD were markedly different and suggest that they exert their protective activities at different sites. Administration of l-NAME and l-TC by a variety of routes did not result in consistent protection against topical vapor challenges of HD in hairless guinea pigs. However, both compounds were effective in preventing the toxicity of HD in primary cultures of hairless guinea pig skin keratinocytes, indicating that species differences were not likely to be responsible for the poor efficacy of these compounds in vivo.     

Utility of MTT assay in three-dimensional cultured human skin model as an alternative for draize skin irritation test: approach using diffusion law of irritant in skin and toxicokinetics-toxicodynamics correlation

Purpose. A cytotoxicity assay using a three-dimensional cultured human skin model, Living Skin Equivalent-high (LSE-high) was evaluated as an alternative to the Draize skin irritation tests using animals. A relation between the cytotoxicity and calculated concentration of an irritant in skin was also evaluated. Methods. Colorimetric thiazoyl blue (MTT) conversion assay and a surfactant, cetylpyridinium chloride (CPC), were selected as a cytotoxicity assay and a model irritant. The fraction of dead cell number in the MTT assay or the Draize irritation score (in vitro and in vivo irritation data, respectively) was treated as a function of CPC concentration in the viable skin of LSE-high and guinea pig. Separately, in vitro permeations of CPC through the LSE-high or excised guinea pig skin were determined to calculate the average concentration of CPC in the viable skin using the Fickian diffusion theory. The obtained relations between the irritation scores and CPC concentration were evaluated by the Emax model (Hill equation). Results. CPC concentration showing 50% irritation (IC ₅₀) was similar for the MTT assay (18.9%) and Draize test (12.3%), and a good relationship (r = 0.981) was observed between the fraction of dead cell number and the Draize score. In contrast, IC ₅₀, 1.32%, for the MTT assay in LSE-high was much lower than that using guinea pig skin. We then corrected the results for the MTT assay using a ratio of IC ₅₀ in guinea pig skin against LSE-high, resulting in a good relation between both MTT results in guinea pig skin and LSE-high. Conclusion. The present results suggest that the MTT assay using LSE-high may be utilized as an alternative for the Draize test in animals for evaluating skin irritation.     

Assessment of the developmental toxicity of nanoparticles in an ex vivo 3D model,the murine limb bud culture system

Abstract

The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is an ex vivo 3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype and Col2a1; Col10a1; Col1a1 triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3–100?nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructures per se or a degradation component released from the complex nanostructure.     

Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells

Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1 mM) for 72 h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6 h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1 mM.     

Inhibitory effects of alpha-arbutin on melanin synthesis in cultured human melanoma cells and a three-dimensional human skin model

We studied the inhibitory effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) on melanogenesis in cultured human melanoma cells, HMV-II, and in a three-dimensional cultured human skin model. alpha-Arbutin showed no inhibitory effect on HMV-II cell growth at a concentration below 1.0 mM. Melanin synthesis in cells treated with alpha-arbutin at 0.5 mM decreased to 76% of that in non-treated cells. The cellular tyrosinase activity of HMV-II cells also significantly decreased, while the expression of its mRNA was not affected. Melanin synthesis in a human skin model was also evaluated by the macro- and microscopic observation of its pigmentation as well as by quantitative measurements of melanin. Treatment of the human skin model with 250 microg of alpha-arbutin did not inhibit cell viability, while melanin synthesis was reduced to 40% of that in the control. These results indicate that alpha-arbutin is an effective and safe ingredient for skin-lightening.     

Probe depth matters in dermal microdialysis sampling of benzoic acid after topical application: an ex vivo study in human skin

Microdialysis (MD) in the skin - dermal microdialysis (DMD) - is a unique technique for sampling of topically as well as systemically administered drugs at the site of action, e.g. sampling of dermatological drug concentrations in the dermis. Debate has concerned the existence of a correlation between the depth of the sampling device - the probe - in the dermis and the amount of drug sampled following topical drug administration. This study evaluates the relation between probe depth and drug sampling using dermal DMD sampling ex vivo in human skin. We used superficial (<1 mm), intermediate (1-2 mm) and deep (>2 mm) positioning of the linear MD probe in the dermis of human abdominal skin, followed by topical application of 4 mg/ml of benzoic acid (BA) in skin chambers overlying the probes. Dialysate was sampled every hour for 12 h and analysed for BA content by high-performance liquid chromatography. Probe depth was measured by 20-MHz ultrasound scanning. The area under the time-versus-concentration curve (AUC) describes the drug exposure in the tissue during the experiment and is a relevant parameter to compare for the 3 dermal probe depths investigated. The AUC(0-12) were: superficial probes: 3,335 ± 1,094 μg·h/ml (mean ± SD); intermediate probes: 2,178 ± 1,068 μg·h/ml, and deep probes: 1,159 ± 306 μg·h/ml. AUC(0-12) sampled by the superficial probes was significantly higher than that of samples from the intermediate and deeply positioned probes (p value <0.05). There was a significant inverse correlation between probe depth and AUC(0-12) sampled by the same probe (p value <0.001, r(2) value = 0.5). The mean extrapolated lag-times (±SD) for the superficial probes were 0.8 ± 0.1 h, for the intermediate probes 1.7 ± 0.5 h, and for the deep probes 2.7 ± 0.5 h, which were all significantly different from each other (p value <0.05). In conclusion, this paper demonstrates that there is an inverse relationship between the depth of the probe in the dermis and the amount of drug sampled following topical penetration ex vivo. The result is of relevance to the in vivo situation, and it can be predicted that the differences in sampling at different probe depths will have a more significant impact in the beginning of a study or in studies of short duration. Based on this study it can be recommended that studies of topical drug penetration using DMD sampling should include measurements of probe depth and that efforts should be made to minimize probe depth variability.