Verónica Martínez

Low Potential Ocular Irritation of Arginine-Based Gemini Surfactants and Their Mixtures with Nonionic and Zwitterionic Surfactants

AbstractPurpose. The aim of this study was to find new biocompatible surfactants and mixtures with low ocular irritant action for application in pharmaceutical formulations and to establish a relationship between their structure and their potential ocular irritant activity. Methods. An alternative method to the Draize in vivo test, based on the adverse effects of surfactants on the cytoplasmic membrane of red blood cell, was used to evaluate the potential ocular irritation of the surfactants. Results. It was found that the hemolytic activity of arginine-based gemini surfactants increased with the aliphatic alkyl chain lengths of the hydrophobic tail. The addition of the surfactant with an alkyl chain length of 10 carbon atoms to cocoamidopropilbetaina (TB), decylglucoside (APG), and Nα-lauroyl-arginine ethyl ester (LAE) increases the hemolytic activity moderately for the mixtures with TB and LAE (1.1- and 1.5-fold, respectively) and strongly for APG (fivefold). Conclusions. The new arginine-based gemini surfactants constitute a suitable alternative to commercial surfactants because of their natural origins, which make them biocompatible and renewable products. Based on their hemolytic activity as an alternative to the Draize test, these new arginine-based gemini surfactants and their mixtures can be classified as mild irritants. This fact constitutes an advantage, especially for pharmaceutical and cosmetic applications.

In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced by Sphingobacterium detergens

The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.

NCTC 2544 and IL-18 production: a tool for the in vitro identification of photoallergens

PURPOSE Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of keratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously shown that IL-18 production in the human keratinocyte cell line NCTC 2455 is a good model for the in vitro identification of contact sensitizers. The purpose of this manuscript is to summarize data obtained in the NCTC 2544 assay as an in vitro model to identify photoallergens and discriminate them from phototoxic chemicals. METHODS The effect of UVA radiation (3.5J/cm(2)) over NCTC 2544 cells irradiated and non irradiated, and treated with increasing concentrations of various compounds including negative compounds (irritants and allergens), ibuprofen and acridine (photoirritants); ketoprofen, promethazine and chlorpromazine (photoirritants/photoallergens); benzophenone, 4-tert-butyl-4-methoxy-dibenzoylmethane, 2-ethylexyl-p-methoxycinnamate and 6-methylcumarin (photoallergens) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay, while a commercially available ELISA kit was used to assess the intracellular content of IL-18. RESULT At no cytotoxic concentrations, allergens and photoallergens induce a dose-related increase in the production of IL-18, whereas irritants and photoirritants failed, indicating the possibility to use the NCTC 2544 assay to identify in vitro photoallergens.

Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumers appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts.

Establishment of an in vitro photoallergy test using NCTC2544 cells and IL-18 production

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of keratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm(2)) by investigating the effect of UVA irradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)-treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxy-dibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals.

New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant, cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1α and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the α-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

Disturbance of erythrocyte lipid bilayer by amino acid-based surfactants

Summary.In an attempt to increase our knowledge regarding the mechanisms of surfactant membrane interaction, we studied the action of several anionic and cationic amino acid-based surfactants on membrane fluidity using fluorescence anisotropy. Anisotropy measurements demonstrated that almost all of the surfactants studied disturbed the external region of the erythrocyte membrane without affecting the core of the bilayer. How the physico-chemical properties and structure of these compounds affect dynamics of the lipid bilayer is discussed in detail.

Role of galloylation and polymerization in cytoprotective effects of polyphenolic fractions against hydrogen peroxide insult.

Byproducts and wastes generated by agricultural, food, and forestry industries contain large amounts of polyphenols, which can be potentially used as sources of natural or semisynthetic antioxidants. This study examined and compared the protection against peroxidative damage induced in erythrocytes and 3T3 cell line of polyphenolic fractions from white grape pomace, pine bark, and witch hazel bark. The work pays special attention to the different degrees of polymerization and galloylation of the extracts to contribute to the understanding of their mechanisms of action. Fractions demonstrated different protections against erythrocyte lipid peroxidation, hemolysis, and 3T3 cytotoxicity caused by H(2)O(2). Galloylation is claimed to be related to antioxidant protective capacity, and it is also responsible for the pro-oxidant effect observed at high doses. The results show that not only the percentage of galloylation but also the degree of polymerization are important modulators of their antioxidant capacity. In this sense, it is crucial that novel polyphenolic fractions were prepared attending a value of 3 for the mean degree of polymerization and did not exceed a 30% of galloylation to reach the highest antioxidant capacity with the lowest cytotoxic effects. For this reason, the grape extracts appear to be the best strategy to fight against hydrogen peroxide cell damage.

Establishment of an in vitro photoassay using THP-1 cells and IL-8 to discriminate photoirritants from photoallergens.

At present, there are no in vivo or in vitro methods developed which has been adopted by regulatory authorities to assess photosensitization induced by chemicals. Recently, we have proposed the use of THP-1 cells and IL-8 release to identify the potential of chemicals to induce skin sensitization. Based on the assumption that sensitization and photosensitization share common mechanisms, the aim of this work was to explore the THP-1 model as an in vitro model to identify photoallergenic chemicals. THP-1 cells were exposed to 7 photoallergens and 3 photoirritants and irradiated with UVA light or kept in dark. Non phototoxic allergens or irritants were also included as negative compounds. Following 24h of incubation, cytotoxicity and IL-8 release were measured. At subtoxic concentrations, photoallergens produced a dose-related increase in IL-8 release after irradiation. Some photoirritants also produced a slight increase in IL-8 release. However, when the overall stimulation indexes of IL-8 were calculated for each chemical, 6 out of 7 photoallergens tested reached a stimulation index above 2, while the entire set of negative compounds had stimulation indexes below 2. Our data suggest that this assay may become a useful cell-based in vitro test for evaluating the photosensitizing potential of chemicals.

Cytoprotective Effects of Polyphenols against Oxidative Damage

Since oxidative stress is a causal factor in many chronic and degenerative pathologies, considerable effort has been made to find antioxidant compounds that prevent the onset of these diseases and counteract their progression. In fact, numerous polyphenols possess radical scavenging/antioxidant activity, especially when studied in cell-free systems as well as in cells in vitro, or even in vivo. Polyphenols—natural compounds with variable phenolic structures—are common in vegetables, fruit, grain, bark, roots, tea and wine. All polyphenols contain one or more aromatic rings with more than one hydroxyl group. They are generally classified into four different groups, depending on the number of phenol rings and chemical groups bound to the rings: flavonoids, phenolic acids, stilbenes, and lignans. This chapter reviews recent work on the capacity of polyphenols to protect cells against oxidative insult.