Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI = 3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI = 150 for CD86 (EC150), and for CD54 (EC200) with LLNA’s EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R = 0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 μg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.     

Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro

The local lymph node assay (LLNA) has been developed to assess skin sensitization, and based on the EC3 value, it can also be used to evaluate allergen potency. Therefore, in the development of in vitro alternatives to the LLNA assay, one should not only consider the hazard identification but also the possibility to classify allergens relatively to their potency.We have recently described a selective release of interleukin-8 (IL-8) by chemical allergens in THP-1 cell line, and identified the activation of p38 mitogen-activated protein kinase (p38 MAPK) as a common pathway. Therefore, the purpose of this study was to expand the number of chemicals tested and to investigate whether IL-8 production and p38 MAPK activation can be used to classify allergens according to their potency.THP-1 cells were exposed to the contact allergens (p-benzoquinone, 2-aminophenol, isoeugenol, diethyl maleate, citral and imidazolidinyl urea), selected according to their potency in the LLNA, and to lactic acid and propylene glycol as non-sensitizers. p38 MAPK activation was evaluated 5–15 min after treatment by FACS analysis, while IL-8 release was assed by ELISA following 24 h of incubation. p38 MAPK was activated by all contact allergens, including the pro-apten isoeugenol, whereas IL-8 release was significantly increased after stimulation with all allergens tested, except for isoeugenol. The failure of isoeugenol may be due to decrease in the stability of IL-8 mRNA. Irritants exposure, as expected, failed to induce both p38 MAPK activation and IL-8 release.A significant correlation between IL-8 release and the LLNA EC₃ was found (Pearson correlation r = 0.743, p = 0.0036, n = 12). On the contrary, the activation of p38 MAPK showed no significant correlation between LLNA data and vigor of p38 MAPK activation.Overall, data presented confirm our previous observations and reveal IL-8 as potential tool not only to identify sensitizers, with the exception of pro-haptens, but also to classify them according to their potency, while p38 MAPK activation allows the identification of all sensitizers, including pro-haptens, but was not useful for potency classification.     

Skin sensitization risk assessment model using artificial neural network analysis of data from multiple in vitro assays

The sensitizing potential of chemicals is usually identified and characterized using in vivo methods such as the murine local lymph node assay (LLNA). Due to regulatory constraints and ethical concerns, alternatives to animal testing are needed to predict skin sensitization potential of chemicals. For this purpose, combined evaluation using multiple in vitro and in silico parameters that reflect different aspects of the sensitization process seems promising.We previously reported that LLNA thresholds could be well predicted by using an artificial neural network (ANN) model, designated iSENS ver.1 (integrating in vitro sensitization tests version 1), to analyze data obtained from two in vitro tests: the human Cell Line Activation Test (h-CLAT) and the SH test. Here, we present a more advanced ANN model, iSENS ver.2, which additionally utilizes the results of antioxidant response element (ARE) assay and the octanol–water partition coefficient (Log P, reflecting lipid solubility and skin absorption). We found a good correlation between predicted LLNA thresholds calculated by iSENS ver.2 and reported values. The predictive performance of iSENS ver.2 was superior to that of iSENS ver.1. We conclude that ANN analysis of data from multiple in vitro assays is a useful approach for risk assessment of chemicals for skin sensitization.     

Development of an in vitro photosensitization assay using human monocyte-derived cells

In this study, with the aim of developing a cell-based in vitro photosensitization assay, we examined whether changes of CD86 and CD54 expression on cells of a human monocytic cell line, THP-1, could be used to assess the photosensitizing potential of chemicals. First, we identified suitable conditions of UV-irradiation (irradiation dose; 5.0 J/cm², irradiation intensity; 1.7 mW/cm²) by investigating the effect of UV-irradiation on CD86 and CD54 expression on untreated or 6-methylcoumarin (a representative photoallergen)-treated THP-1 cells (irradiation method). However, acridine, a representative photo-irritant, augmented CD86 and CD54 expression on THP-1 cells, apparently via induction of reactive oxygen species (ROS). In order to abolish the effect of ROS, we examined CD86 and CD54 expression on THP-1 cells treated with pre-irradiated chemicals (pre-irradiation method). We found that UV-irradiated photoallergens, but not photo-irritants, enhanced CD86 and/or CD54 expression on the THP-1 cells. Finally, based on the results of irradiation, non-irradiation, and pre-irradiation with 18 test chemicals, we built a decision tree, which allows us to distinguish between photoallergens and photo-irritants. We suggest that this system may be useful for in vitro evaluation of the photoallergic potential of chemicals.     

Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis

Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6 h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of cellular response to perfluorooctane sulfonate in human umbilical vein endothelial cells

Perfluorooctane sulfonate (PFOS), a stable fluorosurfactant and global pollutant, can be bioaccumulated. Recently, the possible adverse effects of PFOS on human health have raised concern. In this study, we examined the effects of PFOS on the expression of three important receptors related to endocrine-disrupting chemicals, six inflammatory-related genes, the production of reactive oxygen species (ROS) and the ability of cell adhesion in human umbilical vein endothelial cells (HUVECs). The results demonstrated that the expression of PPARγ and ERα were up-regulated after exposure to PFOS. After a high dose exposure (100 mg/L) for 48 h, the expression of IL-1β, IL-6, COX-2, NOS3, ICAM-1 and P-Selectin were all notably up-regulated, whereas all of them did not show any significant changes after a low dose exposure (50 mg/L) for 24 h. Moreover, the expression of IL-1β, COX-2 and NOS3 were significantly up-regulated, when cells were exposed to 100 mg/L PFOS for 24 h. Meanwhile, the amount of ROS induced by the exposure to high-dose PFOS was significantly increased with increasing incubation times. Furthermore, the adhesion of THP-1 cells onto HUVECs was significantly increased after exposure to 100 mg/L PFOS for 48 h, as observed for the expression of ICAM-1 and P-Selectin. In conclusion, our data suggest that PFOS exposure may play an important role in the vascular inflammatory disorders and endothelial dysfunctions.     

Comparative study on prediction performance of photosafety testing tools on photoallergens

Several testing methods have been established to identify potential phototoxins. The present study was undertaken to clarify the predictive ability of in vitro photosafety assays for photoallergenicity. On the basis of animal and/or clinical photosafety information, 23 photoallergens and 7 non-phototoxic/non-photoallergenic chemicals were selected and subjected to UV/VIS spectral analysis, reactive oxygen species (ROS)/micellar ROS (mROS) assays, and 3T3 neutral red uptake phototoxicity testing (3T3 NRU PT). Of the photoallergens tested, ca. 96% of chemicals had intense UV/VIS absorption with a molar extinction coefficient of over 1000 M^− 1 cm^− 1, and false-positive predictions were made for 3 non-photoallergenic chemicals. In the ROS assay, all photoallergens were found to be potent ROS generators under exposure to simulated sunlight. In the photosafety prediction based on the ROS assay, the individual specificity was 85.7%, and the positive predictivity and negative predictivity were found to be 95.8% and 100%, respectively. Most of the photoirritant chemicals were correctly identified by the 3T3 NRU PT; however, it provided false predictions for ca. 48% of photoallergens. The orders of sensitivity and specificity for photoallergenicity prediction were estimated to be: [sensitivity] ROS assay > UV/VIS absorption ≫ 3T3 NRU PT, and [specificity] 3T3 NRU PT > ROS assay ≫ UV/VIS absorption. Thus, photochemical assays, in particular the ROS assay, can be used for assessment of photoallergenicity, although there were some false-positive predictions.     

Assessment of the skin sensitization potency of eugenol and its dimers using a non-radioisotopic modification of the local lymph node assay

Allergic contact dermatitis is a serious health problem. There is a need to identify and characterize skin sensitization hazards, particularly with respect to relative potency, so that accurate risk assessments can be developed. For these purposes the murine local lymph node assay (LLNA) was developed. Here, we have investigated further a modi fi cation of this assay, non-radioisotopic LLNA, which in place of tritiated thymidine to measure lymph node cell proliferation employs incorporation of 5-bromo-2'-deoxyuridine. Using this method we have examined the skin sensitizing activity of eugenol, a known human contact allergen, and its dimers 2,2'-dihydroxyl-3,3'-dimethoxy-5,5'-diallyl-biphenyl (DHEA) and 4,5'-diallyl-2'-hydroxy-2,3'-dimethoxy phenyl ether (DHEB). Activity in the guinea pig maximization test (GPMT) also measured. On the basis of GPMT assays, eugenol was classified as a mild skin sensitizer, DHEA as a weak skin sensitizer and DHEB as an extreme skin sensitizer. In the non-radioisotopic LLNA all chemicals were found to give positive responses insofar as each was able to provoke a stimulation index (SI) of >or=3 at one or more test concentrations. The relative skin sensitizing potency of these chemicals was evaluated in the non-radioisotopic LLNA by derivation of an ec(3) value (the concentration of chemical required to provoke an SI of 3). The ec(3) values calculated were 25.1% for eugenol, >30% for DHEA and 2.3% for DHEB. Collectively these data suggest that assessments of relative potency deriving from non-radioisotopic LLNA responses correlate well with evaluations based on GPMT results. These investigations provide support for the proposal that the non-radioisotopic LLNA may serve as an effective alternative to the GPMT where there is a need to avoid the use of radioisotopes.     

Formaldehyde solutions in simulated sweat increase human melanoma but not normal human keratinocyte cells proliferation

Our skin is in close contact with clothes most of the time thus risking potentially noxious chemicals contact. One of the potentially harmful manufacturing by-products that can be released by textiles when sweating is formaldehyde, used as an anti-crease treatment. As it is known to be carcinogenic to humans and a potent skin sensitizer, the aim of this study was to investigate its effects on both normal human keratinocytes (HaCaT cells) and on a highly invasive malignant melanoma cell line (SK-MEL-28) in order to contribute to the definition of safety cut-off to be applied to the production processes.Formaldehyde concentrations below the commonly accepted limits (10–50 μM) were obtained by diluting formaldehyde in simulated sweat (UNI EN ISO 105-E04). The effects on cell proliferation were evaluated by cell counting, while ERK pathway activation was evaluated by western blot.Low concentrations of formaldehyde (10 μM) in both acidic and alkaline simulated sweat were able to increase malignant melanoma cell proliferation, while not affecting normal keratinocytes. Melanoma proliferation increase was greater in acidic (pH = 5.5) than in alkaline (pH = 8) conditions. Moreover, formaldehyde stimulation was able to induce ERK pathway activation.The data obtained suggest the need for an even increasing attention to the potentially harmful effects of textile manufacturing by-products.     

Novel approach for classifying chemicals according to skin sensitizing potency by non-radioisotopic modification of the local lymph node assay

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone sensitization test for determining the sensitizing potential of chemicals, and it has the advantage of yielding a quantitative endpoint that can be used to predict the sensitization potency of chemicals. The EC3 has been proposed as a parameter for classifying chemicals according to the sensitization potency. We previously developed a non-radioisotopic endpoint for the LLNA based on 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA), and we are proposing a new procedure to predict the sensitization potency of chemicals based on comparisons with known human contact allergens. Nine chemicals (i.e. diphencyclopropenone, p-phenylenediamine, glutaraldehyde, cinnamicaldehyde, citral, eugenol, isopropyl myristate, propyleneglycol and hexane) categorized as human contact allergen classes 1-5 were tested by the non-RI LLNA with the following reference allergens: 2,4-dinitrochlorobenzene (DNCB) as a class 1 human contact allergen, isoeugenol as a class 2 human contact allergen and alpha-hexylcinnamic aldehyde (HCA) as a class 3 human contact allergen. Consequently, nine test chemicals were almost assigned to their correct allergen class. The results suggested that the new procedure for non-RI LLNA can provide correct sensitization potency data. Sensitization potency data are useful for evaluating the sensitization risk to humans of exposure to new chemical products. Accordingly, this approach would be an effective modification of LLNA with regard to its experimental design. Moreover, this procedure can be applied also to the standard LLNA with radioisotopes and to other modifications of the LLNA.     

Acrylamide-induced mitochondria collapse and apoptosis in human astrocytoma cells

Acrylamide (ACR) can be produced during food processing and has neurotoxic effects in humans. This study aims to determine ACR induced apoptotic responses in human astrocytoma U-1240 MG cells to realize the incurred toxic mechanisms. Under 1 and 2 mM ACR exposure, cell viability decreased as time increased. The increments in sub-G₁ phase were 87.5-fold, and pro-caspase 3 and PARP protein expressions decreased 35% and 54.5% respectively relative to the control after 2 mM ACR treatment. Molecular evidence of Bax/bcl-2 ratio and cytochrome c expression increased 8.86-fold and 6.81-fold as well as pro-caspase 9 decreased 67.8% relative to the control respectively under 2 mM ACR exposure. Trolox, an ROS scavenging agent, attenuated cell death and induced ROS production by 2 mM ACR. The ultrastructure alterations of mitochondria showed marked vesicular matrix compartmentalization and cytoplasmic vacuole formation after 2 mM ACR was treated for 48 h, whereas those treated for 72 h showed chromatin condensation, pyknosis, and swelling. These results indicate long-term exposure to ACR induced mitochondria collapse and finally led to apoptosis. Although 2 mM ACR is higher than average daily intake dosage, workers in chemical industries may be exposed to sufficient doses to entail health risks.     

Reactive oxygen species induced by beauvericin,patulin and zearalenone in CHO-K1 cells

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC₅₀ values obtained on the MTT and NR assays ranged from 0.69 to 79.40 μM and 4.40 to 108.76 μM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H₂-DCFDA was measured. The relative intensity of fluorescence from cells incubated with BEA, PAT and ZEA was approximately 4-, 7- and 4-fold higher in comparison with control cells at 0 min, respectively. Lipid peroxidation induced by ROS generation was assessed using the thiobarbituric acid reactive substances (TBARS) method for 2, 24 and 48 h. The malondialdehyde (MDA) production was increased with BEA and PAT exposure in a concentration- and time-dependent manner. MDA was not increased after 1 and 5 μM ZEA exposures for 2 h, but was slightly increased at 50 μM.In conclusion, PAT was the most cytotoxic mycotoxin to CHO-K1 cells, followed by BEA and ZEA. Mycotoxins reduce cell viability correlated with the increases of ROS generation and MDA formation in concentration- and time-dependent manner. These data suggested that cytotoxicity and ROS generation are mechanisms of mycotoxins mediated toxicity.     

Skin irritation and sensitization: mechanisms and new approaches for risk assessment

Allergic contact dermatitis (ACD) is a common skin disease with a significant social and economic impact. In contrast to irritation, skin sensitization is a response of the adaptive immune system, in which there is a delayed T-cell-mediated allergic response to chemically modified skin proteins. The chemicals that can covalently modify the skin proteins and trigger an allergic reaction are referred to as haptens or sensitizers. Attempts have been made in many countries to reduce the problems of ACD by the implementation of legislations related to skin-sensitizing chemicals, as well as by the early detection and risk assessment of substances with sensitizing properties. For many years, the simple identification of sensitizing chemicals was performed in guinea pig tests. A murine test, the local lymph node assay (LLNA), has been validated as a replacement for the guinea pig tests. Despite the recent introduction of in vitro methods for the identification of sensitizing chemicals, the LLNA results (when coupled with good exposure data) can be used as the starting point for a quantitative risk assessment. The quantitative risk assessment is aimed to identify the safe use thresholds for any potential skin sensitizer.     

Triglyceride-coated nanoparticles: Skin toxicity and effect of UV/IR irradiation on them

Triglyceride (TG) is an important compound on the skin, produced by sebaceous glands, and may change cytotoxicity of different nanoparticles. To date, there is no report about toxicity of nanoparticles coated with TG. On the other hand, the use of ultraviolet (UV) and infrared (IR) with nanoparticles changes nanoparticle cytotoxicity. The combination of nanoparticles with UV or IR is applicable, because it may be used for treatment or detection of local cancers, surface microbial infections and other skin diseases. In this study, different nanoparticles including titanium dioxide, zinc oxide, magnesium oxide, silver, gold, and TG-coated form of these nanoparticles, were added to suspensions of Balb/c skin cells, and then incubated for 24 h at 37 °C. Additionally, TG-coated nanoparticles were treated with UV and IR irradiation for 1 h. Different methods were applied for evaluation of cytotoxicity, including 5-diphenyl-tetrazolium bromide assay, lactate dehydrogenase (LDH) assay, cell metabolic assay, ATP assay, and reactive oxygen species (ROS) generation assay. This research showed that TG-coated nanoparticles had less LDH release and ROS generation with higher cell viability, cell metabolic activity, and ATP level, compared with pristine nanoparticles. In contrast, the combination of UV and IR with TG-coated nanoparticles led to higher LDH release and ROS generation with less cell viability, cell metabolic activity, and ATP level, in comparison with pristine nanoparticles. Overall, pristine metal nanoparticles without irradiation had higher cytotoxicity than metal oxide nanoparticles.     

Evaluation of cytochrome P450 1 (CYP1) and N-acetyltransferase 1 (NAT1) activities in HaCaT cells: Implications for the development of in vitro techniques for predictive testing of contact sensitizers

Xenobiotic metabolizing enzymes like cytochrome P450s and N-acetyltransferase are expressed in keratinocytes and professional antigen-presenting cells. Thus, biotransformation of chemicals applied to the skin can be relevant for their potential to cause skin toxicity and immune responses like allergic contact dermatitis. Considering the keratinocyte cell line HaCaT as a relevant in vitro tool for epidermal biotransformation, we specifically investigated CYP1 (EROD) and N-acetyltransferase 1 (NAT1) activities of three different HaCaT shipments and human primary keratinocytes (NHEK). Solvent treated HaCaT showed EROD levels near the detection limit (0.047 pmol/mg/min), primary keratinocytes (n = 4) were in a range between 0 and 0.76 pmol/mg/min. B[a]P (1 μM) induced EROD activities of 19.0 ± 0.9 pmol/mg/min (n = 11) in HaCaT and 5.8 ± 0.5 pmol/mg/min (n = 4) in NHEK. N-acetylation activities for para-aminobenzoic acid (PABA) were in average 3.4-fold higher in HaCaT compared to NHEK (8 ± 0.5 nmol/mg/min) and varied between the HaCaT shipments (range 12.0–44.5 nmol/mg/min). This was in good agreement with NAT1 promoter P1 dependent mRNA level and N-acetylation of the contact allergen para-phenylenediamine (PPD) under typical cell-based assay conditions. We conclude that HaCaT represent a suitable in vitro model for studying the qualitative contribution of epidermal phase1/phase2 metabolism to toxicological endpoints such as skin sensitization.     

Interlaboratory Evaluation of the Local Lymph Node Assay with 25 Chemicals and Comparison with Guinea Pig Test Data

Abstract

Summary: The murine local lymph node assay (LLNA) has been developed as an alternative method for the identification of skin sensitizing chemicals. Measurement is made of the proliferation of lymphocytes within lymph nodes draining the site of exposure to the test chemical. This report describes a collaborative study in which 25 test chemicals were evaluated in each of four participating laboratories and the results compared with existing data from guinea pig predictive tests. Nineteen chemicals were predicted to be sensitizers in the guinea pig. Of these, 14 were correctly identified in the LLNA (9 by all laboratories and 5 by two or three laboratories). Five chemicals predicted to be contact allergens by guinea pig tests failed to elicit positive LLNA responses. With adoption of a 5 day rather than a 4 day exposure period to the test chemical and administration of maximum soluble test concentrations, positive reactions could be obtained with each of the chemicals initially negative in the LLNA. The LLNA and guinea pig tests were in agreement for all three chemicals predicted to be nonsensitizers in the guinea pig. Positive LLNA responses were obtained with four chemicals (including a re-evaluation of one initially negative in the LLNA) for which guinea pig results were equivocal in three cases and negative in another. These results suggest that the LLNA may provide a rapid and reliable elective prescreen for the identification of contact allergens.     

Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines

Oxidative stress and mitochondrial oxidative damage have been implicated in aging and many common diseases. Mitochondria are a primary source of reactive oxygen species (ROS) in the cell, and are particularly susceptible to oxidative damage. Oxidative damage to mitochondria results in mitochondrial permeability transition (MPT), mitochondrial depolarization, further ROS production, swelling, and release of cytochrome c (cyt c). Cytosolic cyt c triggers apoptosis by activating the caspase cascade. In the present work, we examined the ability of a novel cell-penetrating, mitochondria-targeted peptide antioxidant in protecting against oxidant-induced mitochondrial dysfunction and apoptosis in two neuronal cell lines. Treatment with tert-butyl hydroperoxide (tBHP) for 24 h resulted in lipid peroxidation and significant cell death via apoptosis in both N₂A and SH-SY5Y cells, with phosphatidylserine translocation, nuclear condensation and increased caspase activity. Cells treated with tBHP showed significant increase in intracellular ROS, mitochondrial depolarization and reduced mitochondrial viability. Concurrent treatment with <1 nM SS-31 (D-Arg-Dmt-Lys-Phe-NH₂; Dmt = 2′,6′-dimethyltyrosine) significantly decreased intracellular ROS, increased mitochondrial potential, and prevented tBHP-induced apoptosis. The remarkable potency of SS-31 can be explained by its extensive cellular uptake and selective partitioning into mitochondria. Intracellular concentrations of [³H]SS-31 were 6-fold higher than extracellular concentrations. Studies using isolated mitochondria revealed that [³H]SS-31 was concentrated ∼5000-fold in the mitochondrial pellet. By concentrating in the inner mitochondrial membrane, SS-31 is localized to the site of ROS production, and can therefore protect against mitochondrial oxidative damage and further ROS production. SS-31 represents a novel platform of mitochondria-targeted antioxidants with broad therapeutic potential.     

Combined effects of low levels of palmitate on toxicity of ZnO nanoparticles to THP-1 macrophages

We have recently proposed that the interaction between food components and nanoparticles (NPs) should be considered when evaluating the toxicity of NPs. In the present study, we used THP-1 differentiated macrophages as a model for immune cells and investigated the combined toxicity of low levels of palmitate (PA; 10 or 50 μM) and ZnO NPs. The results showed that PA especially at 50 μM changed the size, Zeta potential and UV–vis spectra of ZnO NPs, indicating a possible coating effect. Up to 32 μg/mL ZnO NPs did not significantly affect mitochondrial activity, intracellular reactive oxygen species (ROS) or release of interleukin 6 (IL-6), but significantly impaired lysosomal function as assessed by neutral red uptake assay and acridine orange staining. The presence of 50 μM PA, but not 10 μM PA, further promoted the toxic effects of ZnO NPs to lysosomes but did not significantly affect other endpoints. In addition, ZnO NPs dose-dependently increased intracellular Zn ions in THP-1 macrophages, which was not significantly affected by PA. Taken together, the results of the present study showed a combined toxicity of low levels of PA and ZnO NPs especially to lysosomes in THP-1 macrophages.