cytotoxicity-assays-with-zebrafish-cell-lines

Cytotoxicity Assays with Zebrafish Cell Lines

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute ...

a-3d-culture-method-spheroids-embryonic-liver-zebrafish-cell

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

Grupo Boticário

The sum of environmental and genetic factors affects the appearance and function of the skin as it ages. The identification of molecular changes that take place during skin aging provides biomarkers and possible targets for therapeutic intervention. Retinoic acid in different formulations has emerged as an alternative to prevent and repair age-related skin damage.

Triple nanoemulsion potentiates the effects of topical treatments with microencapsulated retinol and modulates biological processes related to skin aging.

The decisive role of the epidermis in maintaining body homeostasis prompted studies to evaluate the changes in epidermal structure and functionality over the lifetime. This development, along with the identification of molecular mechanisms of epidermal signaling, maintenance, and differentiation, points to a need for new therapeutic alternatives to treat and prevent skin aging. In addition to recovering age- and sun-compromised functions, proper treatment of the epidermis has important esthetic implications. This study reviews active ingredients capable of counteracting symptoms of epidermal aging, organized according to the regulation of specific age-affected epidermal functions: (1) several compounds, other than retinoids and derivatives, act on the proliferation and differentiation of keratinocytes, supporting the protective barrier against mechanical and chemical insults; (2) natural lipidic compounds, as well as glycerol and urea, are described as agents for maintaining water-ion balance; (3) regulation of immunological pathogen defense can be reinforced by natural extracts and compounds, such as resveratrol; and (4) antioxidant exogenous sources enriched with flavonoids and vitamin C, for example, improve solar radiation protection and epidermal antioxidant activity. The main objective is to provide a functional classification of active ingredients as regulatory elements of epidermal homeostasis, with potential cosmetic and/or dermatological applications. 

Active ingredients against human epidermal aging.

Striae distensae, commonly known as stretch marks, are cutaneous lesions that accompany the hormonal upheavals of the major stages of life: puberty and pregnancy. Stretch marks occur in 90% of women, and they appear as red or purple lines that slowly fade to pale lines on the skin. There have been few studies regarding stretch mark origins, and new preventive and corrective treatments are needed.

Unraveling the molecular and cellular mechanisms of stretch marks.

Menopause is a stage in a woman's life characterized by twelve months of amenorrhoea. This transition happens due to changes in ovarian follicular activity, leading to endocrine, biological and clinical modifications. The main hormones related to these changes and symptoms are oestradiol, LH, FSH, AMH, Inhibin B and GnRH. It is important to point out that the skin is very affected by all these hormone changes, leading to a decrease in collagen content, water content, elasticity, thickness and impacting on all skin layers quality. Aiming to help women go through this period of their lifetimes with a better quality of life, cosmetic and pharmaceutical industries have studied formulations to improve skin quality. In order to study the safety and efficacy of these products, in vitro methods have been developed in order to mimic menopause and aged skin. In addition to that, many clinical methodologies for skin features assessment have also been improved and applied to evaluate the efficacy of treatments or compounds for menopause. Studying and improving skin models and skin evaluation methodologies may help in the identification of therapeutic targets, treatments, drugs and cosmetics along with new insights for future research in the dermatology field.

Revisiting the effects of menopause on the skin: Functional changes, clinical studies, in vitro models and therapeutic alternatives.

Titanium dioxide nanoparticles (TiO NPs) are regularly used in sunscreens because of their photoprotective capacity. The advantage of using TiO on the nanometer scale is due to its transparency and better UV blocking efficiency. Due to the greater surface area/volume ratio, NPs become more (bio)-reactive giving rise to concerns about their potential toxicity. To evaluate the irritation and corrosion of cosmetics, 3D skin models have been used as an alternative method to animal experimentation. However, it is not known if this model is appropriate to study skin irritation, corrosion and phototoxicity of nanomaterials such as TiO NPs. This systematic review (SR) proposed the following question: Can the toxicity of TiO nanoparticles be evaluated in a 3D skin model? This SR was conducted according to the Preliminary Report on Systematic Review and Meta-Analysis (PRISMA). The protocol was registered in CAMARADES and the ToxRTool evaluation was performed in order to increase the quality and transparency of this search. In this SR, 7 articles were selected, and it was concluded that the 3D skin model has shown to be promising to evaluate the toxicity of TiO NPs. However, most studies have used biological assays that have already been described as interfering with these NPs, demonstrating that misinterpretations can be obtained. This review will focus in the possible efforts that should be done in order to avoid interference of NPs with biological assays applied in 3D  culture.

Toxicity Evaluation of TiO Nanoparticles on the 3D Skin Model: A Systematic Review.

The continuous search for natural products that attenuate age-related losses has increasingly gained notice; among them, those applicable for skin care have drawn significant attention. The bioester generated from the Chenopodium quinoa's oil is a natural-origin ingredient described to produce replenishing skin effects. With this as motivation, we used shotgun proteomics to study the effects of quinoa bioester on human reconstructed epidermis tridimensional cell cultures after 0, 3, 6, 12, 24, and 48 h of exposure. Our experimental setup employed reversed-phase nano-chromatography coupled online with an Orbitrap-XL and PatternLab for proteomics as the data analysis tool. Extracted ion chromatograms were obtained as surrogates for relative peptide quantitation. Our findings spotlight proteins with increased abundance, as compared to the untreated cell culture counterparts at the same timepoints, that were related to preventing premature aging, homeostasis, tissue regeneration, protection against ultraviolet radiation and oxidative damage.

Proteomics reveals that quinoa bioester promotes replenishing effects in epidermal tissue.

The safety assessment of cosmetic products is based on the safety of the ingredients, which requires information on chemical structures, toxicological profiles, and exposure data. Approximately 6% of the population is sensitized to cosmetic ingredients, especially preservatives and fragrances. In this context, the aim of this study was to perform a hazard assessment and risk characterization of benzalkonium chloride (BAC), benzyl alcohol (BA), caprylyl glycol (CG), ethylhexylglycerin (EG), chlorphenesin (CP), dehydroacetic acid (DHA), sodium dehydroacetate (SDH), iodopropynyl butylcarbamate (IPBC), methylchloroisothiazolinone and methylisothiazolinone (MCI/MIT), methylisothiazolinone (MIT), phenoxyethanol (PE), potassium sorbate (PS), and sodium benzoate (SB). Considering the integrated approaches to testing and assessment (IATA) and weight of evidence (WoE) as a decision tree, based on published safety reports. The hazard assessment was composed of a toxicological matrix correlating the toxicity level, defined as low (L), moderate (M), or high (H) and local or systemic exposure, considering the endpoints of skin sensitization, skin irritation, eye irritation, phototoxicity, acute oral toxicity, carcinogenicity, mutagenicity/genotoxicity, and endocrine activity. In a risk assessment approach, most preservatives had a margin of safety (MoS) above 100, except for DHA, SDH, and EG, considering the worst-case scenario (100% dermal absorption). However, isolated data do not set up a safety assessment. It is necessary to carry out a rational risk characterization considering hazard and exposure assessment to estimate the level of risk of an adverse health outcome, based on the concentration in a product, frequency of use, type of product, route of exposure, body surface location, and target population.

Integrated approaches to testing and assessment as a tool for the hazard assessment and risk characterization of cosmetic preservatives.

Dermal contact is the main route of exposure for most cosmetics; however, inhalation exposure could be significant for some formulations (e.g., aerosols, powders). Current cosmetic regulations do not require specific tests addressing respiratory irritation and sensitisation, and despite the prohibition of animal testing for cosmetics, no alternative methods have been validated to assess these endpoints to date. Inhalation hazard is mainly determined based on existing human and animal evidence, read-across, and extrapolation of data from different target organs or tissues, such as the skin. However, because of mechanistic differences, effects on the skin cannot predict effects on the respiratory tract, which indicates a substantial need for the development of new approach methodologies addressing respiratory endpoints for inhalable chemicals in general. Cosmetics might present a particularly significant need for risk assessments of inhalation exposure to provide a more accurate toxicological evaluation and ensure consumer safety. This review describes the differences in the mechanisms of irritation and sensitisation between the skin and the respiratory tract, the progress that has already been made, and what still needs to be done to fill the gap in the inhalation risk assessment of cosmetic ingredients.

Beyond dermal exposure: The respiratory tract as a target organ in hazard assessments of cosmetic ingredients.

Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3-5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.

Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods.

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed.

Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients.

Photoaging is the process by which ultraviolet rays gradually induce clinical and histological changes in the skin through the production and organization of biological molecules, such as elastin, which is critical to skin strength and elasticity. After exposure to radiation, elastin may undergo alternative mRNA splicing, resulting in modified proteins that contribute to the formation of aging characteristics, such as solar elastosis. The present work aimed to study two different forms of elastin under these conditions: normal elastin and elastin that had been altered in exon 26A.

Modulation of Photoaging-Induced Cutaneous Elastin: Evaluation of Gene and Protein Expression of Markers Related to Elastogenesis Under Different Photoexposure Conditions.

Aluminum chlorohydrate (ACH) is a major aerosol component frequently used as the active ingredient in antiperspirants, and in vivo studies have raised a concern about its inhalation toxicity. Still, few studies have addressed its effects on the human respiratory tract. Therefore, we developed a study on ACH inhalation toxicity using an in vitro human alveolar cell model (A549 cells) with molecular and cellular markers of oxidative stress, immunotoxicity, and epigenetic changes. The chemical characterization of ACH suspensions indicated particle instability and aggregation; however, side-scatter analysis demonstrated significant particle uptake in cells exposed to ACH. Exposure of A549 cells to non-cytotoxic concentrations of ACH (0.25, 0.5, and 1 mg/ml) showed that ACH induced reactive oxygen species. Moreover, ACH upregulated TNF, IL6, IL8, and IL1A genes, but not the lncRNAs NEAT1 and MALAT1. Finally, no alterations on the global DNA methylation pattern (5-methylcytosine and 5-hydroxymethylcytosine) or the phosphorylation of histone H2AX (γ-H2AX) were observed. Our data suggest that ACH may induce oxidative stress and inflammation on alveolar cells, and A549 cells may be useful to identify cellular and molecular events that may be associated with adverse effects on the lungs. Still, further research is needed to ensure the inhalation safety of ACH.

ArticleTotal : 2	Year
Publication title Cited by 7	2023
Publication title Cited by 4	2023

Article	Year
Triple nanoemulsion potentiates the effects of topical treatments with microencapsulated retinol and modulates biological processes related to skin aging. Anais brasileiros de dermatologia\| PubMed ID: 24474102	2013
Active ingredients against human epidermal aging. Ageing research reviews\| PubMed ID: 24675046	2014
Unraveling the molecular and cellular mechanisms of stretch marks. Journal of cosmetic dermatology\| PubMed ID: 31131982	2020
Revisiting the effects of menopause on the skin: Functional changes, clinical studies, in vitro models and therapeutic alternatives. Mechanisms of ageing and development\| PubMed ID: 31811831	2020
Toxicity Evaluation of TiO Nanoparticles on the 3D Skin Model: A Systematic Review. Frontiers in bioengineering and biotechnology\| PubMed ID: 32587852	2020
Proteomics reveals that quinoa bioester promotes replenishing effects in epidermal tissue. Scientific reports\| PubMed ID: 33173110	2020
Integrated approaches to testing and assessment as a tool for the hazard assessment and risk characterization of cosmetic preservatives. Journal of applied toxicology : JAT\| PubMed ID: 33624850	2021
Beyond dermal exposure: The respiratory tract as a target organ in hazard assessments of cosmetic ingredients. Regulatory toxicology and pharmacology : RTP\| PubMed ID: 34139277	2021
Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods. Ecotoxicology (London, England)\| PubMed ID: 34379241	2021
Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients. Regulatory toxicology and pharmacology : RTP\| PubMed ID: 34389358	2021
Modulation of Photoaging-Induced Cutaneous Elastin: Evaluation of Gene and Protein Expression of Markers Related to Elastogenesis Under Different Photoexposure Conditions. Dermatology and therapy\| PubMed ID: 34648146	2021
In vitro evaluation of the inhalation toxicity of the cosmetic ingredient aluminum chlorohydrate. Journal of applied toxicology : JAT\| PubMed ID: 35883269	2022

Marcio Lorencini

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