Miriam Oron

Novel chemiluminescence-inducing cocktails, part II: Measurement of the anti-oxidant capacity of vitamins, thiols, body fluids, alcoholic beverages and edible oils

Using two luminescence-inducing cocktails, two distinct patterns of inhibition of light by different anti-oxidants have been identified, comprising Group A, in which a complete inhibition of light emission which is then followed by re-emergence of light, forming apparent S-shaped curves or similar shapes. This light pattern is induced by the “classical” anti-oxidants, ascorbate, vitamin E, uric acid, thiols, deferoxamine, as well as by anti-oxidant agents present in plasma, saliva, urine and in extracts derived from black coffee, and Group B, in which a gradually emerging “mound”-shaped pattern of light was seen with extracts from the Tibetan plant mixture PADMA-28, elderberry (Sambucol), grape seeds, green and black teas, apple, parsimony, red wines, edible oils and SOD. While the results with the Group A agents point to the presence of probably a single, major, anti-oxidants relatively sensitive to oxidation, Group B agents probably include a mixture of anti-oxidants which are more resistant to oxidation. It was also shown that agents from Group B could protect agents from Group A against consumption by the oxidants generated by the cocktails. It is proposed that these simple to use cocktails which probably generate a multiplicity of oxidants mimicking those generated by activated phagocytes, can rapidly assess the total anti-oxidant capacities (TAOC) in body fluids derived from patients suffering of excessive oxidative stress. Also, this technique may be useful in determining the content of dietary anti-oxidants recommended as supplements to enhance the resistance against excessive oxidation of lipids.

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin

Background:  Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known.

Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: Influence of synthesis method, crystalline structure, size and additive

Abstract Increasing the production and applications of TiO2 nanoparticles (NPs) has led to grow concerns about the consequences for the environment. In this study, we investigated the effects of a set of TiO2 NPs on the viability of mussel hemocytes and gill cells using neutral red and thiazolyl tetrazolium bromide assays. For this, we compared the cytotoxicity of TiO2 NPs (0.1–100 mg Ti/L) produced by different techniques: rutile NPs (60 nm) produced by milling and containing disodium laureth sulfosuccinate (DSLS), rutile NPs (10, 40 and 60 nm) produced by wet chemistry and anatase/rutile NPs (∼100 nm) produced by plasma synthesis. The commercially available P25 anatase/rutile NPs (10–20 nm) were also tested. Exposures were performed in parallel with their respective bulk forms and the cytotoxicity of the additive DSLS was also tested. Z potential values in distilled water indicated different stabilities depending on the NP type and all NPs tested formed agglomerates/aggregates in cell culture media. In general, TiO2 NPs showed a relatively low and dose-dependent toxicity for both cell models with the two assays tested. NPs produced by milling showed the highest effects, probably due to the toxicity of DSLS. Size-dependent toxicity was found for NPs produced by wet chemistry (10 nm > 40 nm and 60 nm). All TiO2 NPs tested were more toxic than bulk forms excepting for plasma produced ones, which were the least toxic TiO2 tested. The mixture bulk anatase/rutile TiO2 was more toxic than bulk rutile TiO2. In conclusion, the toxicity of TiO2 NPs varied with the mode of synthesis, crystalline structure and size of NPs and can also be influenced by the presence of additives in the suspensions.

Novel chemiluminescence-inducing cocktails, part I: The role in light emission of combinations of luminal with SIN-1, selenite, albumin, glucose oxidase and Co2+

It is known that many agents influence the capacity of cells to produce reactive oxygen species. However, assaying these agents, both those that stimulate and those that inhibit reactive oxygen production, can be complicated and time consuming. Here, a method is described in which two different cocktails are employed to stimulate luminol-dependent chemiluminescence (LDCL). These cocktails are comprised of luminol, with either sodium selenite [IV] (SEL) or tellurite [IV] (TEL) (where IV and VI refer to the 4+ or 6+ oxidation state of selenium or tellurium salts, respectively), morpholinosidonimine (SIN-1), serum albumin and Co2+, called the SIN-1a (with selenite) and SIN1b (with tellurite) cocktails, respectively; or luminol with glucose oxidase (GO), sodium selenite [IV] and Co2+, called the GO cocktail. The cocktails functioned best in Hank’s balanced salt solution (HBSS) containing 1% glucose at pH 7.4, incubated at approximately 22°C. Within 30–60 s there was a burst of luminescence, which lasted for 7–10 min. In 100% ethanol, the SIN-1 cocktails also generated LDCL to 70% of that produced in HBSS. Neither selenite [VI], seleno-cystine, seleno-methionine, nor the selenium-containing drug, ebselen, could replace SEL. Moreover, the effects of the NO-donor, SIN-1, could not be replicated by the oxyradical generators, xanthine-xanthine oxidase or hypochlorous acid. Only low levels of luminescence were generated by combinations of the peroxyl radical generator, 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH) with either SEL or TEL. It is suggested that light emission induced by the SIN1 cocktail results from the oxidation of SEL [IV] to the [VI] state, possibly due to the generation of mixtures of superoxide, peroxide, peroxynitrite and also of unidentified oxidant species, catalyzed by CoCo2+. However, the involvement of hydroxyl radicals in LDCL could not be confirmed by use of either dimethyl thiourea or by electron spin resonance (ESR). LDCL induced by the two cocktails is strongly reduced by phosphates, EDTA, deferoxamine, CuCo2+, MnCo2+, as well as by the “classical” antioxidants superoxide dismutase (SOD), ascorbate, vitamin E, uric acid or thiols. It is suggested that these chemiluminescence cocktail systems can be used to determine the total anti-oxidant capacities of biological fluids and commercially available anti-oxidants.

Evaluation of topically applied copper(II) oxide nanoparticle cytotoxicity in human skin organ culture.

The increasing use of nano-sized materials in our environment, and in many consumer products, dictates new safety concerns. In particular, adequate experimental models are needed to evaluate skin toxicity of metal oxide ions, commonly found in cosmetic and dermatologic preparations. We have addressed the biological effects of topically applied copper oxide (CuO) nanoparticles in human skin organ cultures, using light and electron microscopy, and biochemical tests. Nanoparticles were more toxic than micro-sized particles, and their effects were stronger when supplied in growth medium than in topical application. Still topically applied CuO nanoparticles induced inflammatory cytokine secretion and necrosis, especially in epidermis deprived of its protective cornea. Since nanoparticle penetration was not seen, we propose that they may adhere to skin surface, react with the local acidic environment, and generate soluble ions that make their way to inner sites. This work illustrates the abilities of skin organ culture to evaluate the biological effects of topically-applied materials on skin in vitro.

Non-invasive skin biomarkers quantification of psoriasis and atopic dermatitis: cytokines, antioxidants and psoriatic skin auto-fluorescence.

BACKGROUND Psoriasis and atopic dermatitis (AD) are challenging to treat due to the absence of suitable monitoring procedure and their recurrences. Alteration of skin hydrophilic biomarkers (SHB) and structural elements occur in both disorders and may possess a distinct profile for each clinical condition. OBJECTIVE To quantify skin cytokines and antioxidants non-invasively in psoriatic and in AD patients and to evaluate skin auto-fluorescence in psoriatic patients. METHODS A skin wash sampling technique was utilized to detect the expression of SHB on psoriatic and AD patients and healthy controls. Inflammatory cytokine (TNFα, IL-1α and IL-6) levels, total antioxidant scavenging capacity and uric acid content were estimated. Additionally, measurement of the fluorescent emission spectra of tryptophan moieties, collagen cross-links and elastin cross-links were performed on psoriatic patients and healthy controls. RESULTS Our findings demonstrate significant alterations of the SHB levels among psoriasis, AD and healthy skin. Differences were also observed between lesional and non-lesional areas in patients with psoriasis and AD. Ultra-structural changes were found in psoriatic patients both in lesional and non-lesional areas. CONCLUSION Employing non-invasive measurements of skin wash sampling and skin auto-fluorescence might serve as complementary analysis for improved diagnosis and treatment of psoriasis and AD. Furthermore, they may serve as an additional monitoring tool for various diseases, in which skin dysfunction is involved.

Cytotoxicity of Au, ZnO and SiO2 NPs using in vitro assays with mussel hemocytes and gill cells: Relevance of size, shape and additives

Abstract Metal-bearing nanoparticles (NPs) possess unique physico-chemical characteristics that make them useful for an increasing number of industrial products and applications, but could also confer them a higher toxicity due to their higher reactivity compared to bulk forms of the same materials. There is a considerable interest in the use of in vitro techniques in environmentally relevant species, such as marine mussels, to evaluate NPs toxicity. In the present work, mussel hemocytes and gill cells were used to assess the potential toxic effects of Au, ZnO and SiO2 NPs with different sizes and shapes in parallel with their respective ionic and bulk forms and additives used in the NPs preparations. Cytotoxicity (neutral red and MTT assays) was screened at a wide range of concentrations, and LC50 values were calculated. Uptake of fluorescently labeled SIO2 NPs of 27 nm by hemocytes was also investigated. Au, ZnO and SiO2 NPs were less toxic than the corresponding ionic forms but more toxic than the bulk forms. ZnO NPs were the most toxic NPs tested which could be related with their capacity to release free ions. SiO2 NPs were not taken up by hemocytes and were not toxic to either hemocytes or gill cells. Size-dependent toxicity was found for Au NPs. Shape influenced the cytotoxicity of ZnO NPs. Finally, the presence of the additives Na-citrate and Ecodis P90 contributed to the toxicity of Au and ZnO NPs, respectively. As a general conclusion, solubility appears to play a key role in NPs toxicity to mussel cells.

Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles

Abstract The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO2 nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO2 NPs (60 and 180 nm) or bulk TiO2, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO2 NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO2 NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO2 NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO2 NPs although more markedly after exposure to TiO2-60 NPs. Exposure to bulk TiO2 and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO2 and DSLS.

Low molecular weight antioxidants released from the skin's epidermal layers: an age dependent phenomenon in the rat

Skin is one of the tissues most exposed to oxidative stress both from endogenous and exogenous sources. Therefore, it can be speculated that skin possesses an extremely efficient antioxidant defense mechanism, particularly in its epidermal layers. The present study shows that human and rat skins possess different and unique reducing antioxidant profiles. These reducing antioxidants can be washed out into the surrounding environment. Non-invasive measurements indicated that skin releases low molecular weight antioxidants (LMWA) from its epidermal layers. Cyclic voltammetry measurements have shown that rat skin releases three major groups of reducing antioxidants at peak potentials of 476 and 889 and 1044 mV while human skin releases two major groups at peak potentials of 779 and 1068 mV. In rat, the overall concentrations of the LMWA secreted decreased significantly with age. The major components of the LMWA composing the first anodic wave in rats were identified as uric acid and ascorbic acid. Uric acid and other as yet uncharacterized LMWA, but not ascorbic acid, were released in human skin. Differences in the ability to release high levels of uric acid among species were well correlated with their metabolic rates. It is suggested that in rat the released LMWA may serve as a possible marker for aging of the skin.

Effects of metal-bearing nanoparticles (Ag, Au, CdS, ZnO, SiO2) on developing zebrafish embryos

Due to the increasing commercialization of consumer and industrial products containing nanoparticles (NPs), an increase in the introduction of these materials into the environment is expected. NP toxicity to aquatic organisms depends on multiple biotic and abiotic factors, resulting in an unlimited number of combinations impossible to test in practice. The zebrafish embryo model offers a useful screening tool to test and rank the toxicity of nanomaterials according to those diverse factors. This work aims to study the acute and sublethal toxicity of a set of metal-bearing NPs displaying different properties, in comparison to that of the ionic and bulk forms of the metals, in order to establish a toxicity ranking. Soluble NPs (Ag, CdS and ZnO) showed the highest acute and sublethal toxicity, with LC50 values as low as 0.529 mg Ag l(-1) for Ag NPs of 20 nm, and a significant increase in the malformation prevalence in embryos exposed to 0.1 mg Cd l(-1) of CdS NPs of ∼4 nm. For insoluble NPs, like SiO2 NPs, acute effects were not observed during early embryo development due to the protective effect of the chorion. But effects on larvae could be expected, since deposition of fluorescent SiO2 NPs over the gill lamella and excretion through the intestine were observed after hatching. In other cases, such as for gold NPs, the toxicity could be attributed to the presence of additives (sodium citrate) in the NP suspension, as they displayed a similar toxicity when tested separately. Overall, the results indicated that toxicity to zebrafish embryos depends primarily on the chemical composition and, thus, the solubility of the NPs. Other characteristics, such as size, played a secondary role. This was supported by the observation that ionic forms of the metals were always more toxic than the nano forms, and bulk forms were the least toxic to the developing zebrafish embryos.