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New approach methodologies (NAMs), including in vitro toxicology methods such as human cells from simple cell cultures to 3D and organ-on-a-chip models of human lung, intestine, liver, and other organs, are challenging the traditional “norm” of current regulatory risk assessments. Uncertainty Factors continue to be used by regulatory agencies to account for perceived deficits in toxicology data. With the expanded use of human cell NAMs, the question “Are uncertainty factors needed when human cells are used?” becomes a key topic in the development of 21st-century regulatory risk assessment. M.D., PhD, the coauthor of an article detailing uncertainty factors within the U.S. EPA, and L.E., PhD., Executive Vice President, Science, Emulate, who is involved in developing organ-on-a-chip models, debated the topic. One important outcome of the debate was that in the case of in vitro human cells on a chip, the interspecies (animal to human) uncertainty factor of 10 could be eliminated. However, in the case of the intraspecies (average human to sensitive human), the uncertainty factor of 10, additional toxicokinetic and/or toxicodynamic data or related information will be needed to reduce much less eliminate this factor. In the case of other currently used uncertainty factors, such as lowest observable adverse effect level to no-observed adverse effect level extrapolation, missing important toxicity studies, and acute/subchronic to chronic exposure extrapolation, additional data might be needed even when using in vitro human cells. Collaboration between traditional risk assessors with decades of experience with in vivo data and risk assessors working with modern technologies like organ chips is needed to find a way forward.

Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma.

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma.

Different models of reconstructed human epidermis (RHE) are currently validated to assess skin irritation in vitro and ultimately to the animal replacement of the Draize test. The development of a new RHE model is a challenge for many laboratories, representing a potential gain of autonomy and improvement of technological knowledge. The Organization for Economic Co-operation and Development (OECD) encourages the development of new models and, for this purpose, offers a thorough guideline on quality control parameters (OECD TG 439 performance standards). This work aimed to develop an RHE model (i.e. USP-RHE) for in vitro skin irritation assays, following the OECD TG 439. The developed model presents a well-differentiated epidermis similar to the Validated Reference Methods (VRM) and to native human epidermis. Quality parameters, i.e. optical density of negative control, tissue integrity and barrier function, were similar to VRM and in accordance with OECD TG 439. Moreover, the USP-RHE model was shown to have 85,7% of specificity (6/7), 100% of sensitivity (6/6) and 92,3% of accuracy (12/13) when compared to in vivo UN GHS classification. The within-laboratory reproducibility was 92.3% (12/13). Thus, we demonstrated that USP-RHE model attends to all OECD TG 439 performance standards and is ready to be used by private and public laboratories and companies for future validation studies.

Made available in DSpace on 2018-11-26T17:55:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The Acid Black 210 dye is one of the most used black dyes by the leather industry. This compound contains three azo groups in its chemical structure, and has been quoted as a non-regulated dye with toxicological concern, since it could generate carcinogenic aromatic amines. The objective of this study was to perform the ecotoxicological risk assessment of this dye through testing its toxicity in vitro and in vivo with the Ames test, the Comet assay, the Daphnia similis test, and the zebrafish embryo acute toxicity test. Moreover, we evaluated the presence of this dye in environmental samples related with a tannery industry. All the tests performed were negative, with the exception of the Ames test with the Salmonella typhimurium TA98 strain, which resulted in a low mutagenic potency. Due to the low concentrations of the Acid Black 210 dye found in tannery effluents, and the high concentrations where any toxic activity is occasionally described, we concluded that this dye is safe from the ecotoxicological point of view in the areas evaluated and in the light of the current knowledge. (C) 2016 Elsevier Ireland Ltd. All rights reserved. Univ Sao Paulo, Lab Environm Toxicol, Sch Pharmaceut Sci Ribeirao Preto, BR-14040903 Ribeirao Preto, SP, Brazil Univ Estadual Paulista, Inst Chem, BR-14800060 Araraquara, SP, Brazil Univ Sao Paulo, Sch Pharmaceut Sci, Skin Biol Lab, BR-05508000 Sao Paulo, SP, Brazil Univ Estadual Paulista, Inst Chem, BR-14800060 Araraquara, SP, Brazil FAPESP: 2008/10449-7 FAPESP: 2013/09317-7 FAPESP: 2013/06172-8 FAPESP: 2013/24340-5

Background Inverse associations have been observed between memory performance and blood concentrations of cadmium (Cd) and lead (Pb). Low antioxidant cell activity has also been linked to decline in memory due to aging. However, it has not yet been established whether the heavy metal-memory relationship is mediated by differences in antioxidant activity. Methods We examined Cd and Pb levels, as well as oxidative stress parameters, in blood samples from 125 older adults (age range 50–82 years). The Counting Span Test (CST) was used to evaluate working memory capacity (WMC). The Monte Carlo Method for Assessing Mediation (MCMAM) was used to analyze the mediation role of antioxidant activity in the heavy metals-memory association. Results High blood Cd (BCd) concentration alone, and in combination with elevated blood Pb (BPb) concentration, was associated with poor WMC (p ≤ 0.001) and low enzymatic antioxidant defenses (p ≥ 0.006). The variance in WMC accounted for by BCd or by BCd combine with BPb was 20.6% and 18.6%, respectively. The MCMAM revealed that the influence of BCd and BPb concentrations on WMC was mediated by low antioxidant capacity (confidence interval - CI: 0.072 to − 0.064 for BCd; CI: − 0.062 to − 0.045 for BPb). Conclusion These findings showed Pb and Cd blood concentration in older adults, even at levels below the current recommended threshold, was negatively associated with WMC and that this relationship may be partly mediated by low antioxidant defenses. Knowledge on the environmental factors that negatively influence brain and cognition during aging can help inform public policy strategies to prevent and control the adverse effects of environmental contaminant exposure during aging.

NRAS-mutations arise in 15-20% of all melanomas and are associated with aggressive disease and poor prognosis. Besides, the treatment for NRAS-mutant melanoma are not very efficient and is currently limited to immune checkpoints inhibitors or aggressive chemotherapy. 4-nerolidylcathecol (4-NC), a natural product extracted from Pothomorphe umbellata, induces apoptosis in melanoma cells by ROS production, DNA damage and increased p53 expression, in addition to inhibiting invasion in reconstructed skin. Moreover, 4-NC showed cytotoxicity in BRAF/MEKi-resistant and naive melanoma cells by Endoplasmic Reticulum (ER) stress induction in vitro. We evaluated the in vivo efficacy and the systemic toxicity of 4-NC in a NRAS-mutant melanoma model. 4-NC was able to significantly suppress tumor growth 4-fold compared to controls. Cleaved PARP and p53 expression were increased indicating cell death. As a proof of concept, MMP-2 and MMP-14 gene expression were decreased, demonstrating a possible role of 4-NC in melanoma invasion inhibition. Toxicological analysis indicated minor changes in the liver and bone marrow, but this toxicity was very mild when compared to other proteasome inhibitors and ER stress inductors already described. Our data indicate that 4-NC can counteract melanoma growth in vivo with minor adverse effects, suggesting further investigation as a potential NRAS-mutant melanoma treatment.

A receptor-independent (RI) four-dimensional structure-activity relationship (4D-QSAR) formalism was applied to a set of sixty-four β-N-biaryl ether sulfonamide hydroxamate derivatives, previously reported as potent inhibitors against matrix metalloproteinase subtype 9 (MMP-9). MMP-9 belongs to a group of enzymes related to the cleavage of several extracellular matrix components and has been associated to cancer invasiveness/metastasis. The best RI 4D-QSAR model was statistically significant (N=47; r(2) =0.91; q(2) =0.83; LSE=0.09; LOF=0.35; outliers=0). Leave-N-out (LNO) and y-randomization approaches indicated the QSAR model was robust and presented no chance correlation, respectively. Furthermore, it also had good external predictability (82 %) regarding the test set (N=17). In addition, the grid cell occupancy descriptors (GCOD) of the predicted bioactive conformation for the most potent inhibitor were successfully interpreted when docked into the MMP-9 active site. The 3D-pharmacophore findings were used to predict novel ligands and exploit the MMP-9 calculated binding affinity through molecular docking procedure.

Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH4Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH4Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation.

Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Mutations in the MAPK (Ras-Raf-MEK-ERK) pathway is closely linked to the lack of control of cell proliferation. Especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent inhibitors of v-Raf murine sarcoma viral oncogene homolog B (BRAFi) and mitogen-activated protein kinase kinase (MEKi). Very high rates of response have been achieved, but most patients are relapsed due to the development of resistance, justifying the constant search for new therapeutic compounds. Early results from our group indicated that 4-nerolidylcatechol (4-NC), a catechol compound extracted from Pothomorphe umbellata, induces DNA damage, ROS production, increased p53 expression culminating in apoptosis in melanoma but with no data regarding the 4-NC effects in cells resistant to BRAFi or MEKi. Therefore, here we evaluated the role of 4-NC alone or in combination with BRAFi/MEKi in resistant melanoma cells. Double-resistant cells were generated and characterized by MAPK pathway reactivation. 4-NC alone or in combination (30 μM) with MAPK inhibitors was cytotoxic, inhibited colony formation and decreased invasiveness in two and three-dimensional cell culture models of treatment-naive, BRAFi-resistant and BRAF/MEKi double-resistant melanoma cells. Apoptosis induction was demonstrated in resistant and double-resistant melanoma cell lines after 4-NC treatments. 4-NC showed important ability to induce apoptosis via Endoplasmatic Reticulum (ER) stress and specifically BiP and CHOP that had increased protein expression in all melanoma cell lines proving to be part of the ER stress pathway activation. CHOP knockdown slightly but enough increases cellular viability following 4-NC treatment indicating that apoptosis observed is partially dependent on CHOP. In summary, we show that 4-NC is a compound with activity against cutaneous melanoma, including resistant cells to clinically approved therapies.

Knowing the damage that particulate matter (PM) can cause in skin is important for tightly controlling the release of air pollutants and preventing more serious diseases. This study investigates if such alterations are present in reconstructed human epidermis exposed to coarse air PM. Exposure of reconstructed human epidermis to increasing concentrations (2.2, 8.9, and 17.9 μg/cm2) of standard urban PM over time led to decreased cell viability at 48 hours. The barrier function was shown to be compromised by 24 hours of exposure to high doses (17.9 μg/cm2). Morphological alterations included cytoplasm vacuolization and partial loss of epidermal stratification. Cytokeratin 10, involucrin, loricrin, and filaggrin protein levels were significantly decreased. We confirmed an inflammatory process by IL-1α release and found a significant increase in AQP3 expression. We also demonstrated changes in NOTCH1 and AhR expression of epidermis treated with coarse air PM. The use of hydrogen peroxide altered AQP3 and NOTCH1 expression, and the use of N-acetyl-L-cysteine altered NOTCH1 expression, suggesting that this is a redox-dependent process. These results demonstrate that coarse air PM induces dose-dependent inflammatory response and alterations in protein markers of differentiation and water transport in the epidermis that could ultimately compromise the structural integrity of the skin, promoting or exacerbating various skin diseases.

Gliomas account for the majority of primary malignant brain tumors and present invasive behavior into adjacent healthy tissue. While 4-NC had previously shown to induce apoptotic cell death in a melanoma model, for the glioma model described in this paper 4-NC is cytotoxic for the cells with the induction of the autophagic pathway. Trypan blue exclusion assay showed that 4-NC was cytotoxic in a dose-dependent manner for A172 and T98G cell lines. IC10 and IC50 values were at 32 µM and 41 µM for A172 and T98G respectively. Inhibition of cell proliferation was observed by total cell counts and by cell cycle analysis by flow cytometry, with cell cycle arrest of A172 and T98G cell lines respectively in the G1/G0 and S phases of the cell cycle. 4-NC induced up-regulation of autophagic pathways, as shown by immunoblotting for LC3-I/II, Real-Time PCR for ATG-7 and Beclin-1 genes, and by fluorescence microscopy observation of autophagic vacuoles in cells transfected with GFP-LC3 and electron microscopy. Glioma cells concomitantly treated with 4-NC and 3-MA, an inhibitor of the autophagic process, are more sensible to cell death, suggesting that autophagy protects the cells from the action of 4-NC.

Allergic and irritant skin reactions caused by topical exposure to permanent hair dyes are a common problem. For regulatory and ethnical purposes, it is required to perform chemical safety assessment following the replacement, reduction, and refinement of animal testing (312s). Permanent hair dyes are formed by a mixture of ingredients that vary from low to extreme skin sensitizing potency and that inter-react to form unknown by-products. Because of the complex reaction, this cytotoxic mechanism has not yet been elucidated and is the subject of this study. Here, we topically exposed p-phenylenediamine (PPD), Resorcinol (RES), Hydrogen Peroxide (H2O2) alone or as a mixture to RhE and evaluated parameters related to skin irritation such as epidermal' viability, keratinocytes damage, barrier loss and IL-1 alpha. Our data indicates that ingredients tested alone did not lead to an increase of cytotoxic parameters related to skin irritation. However, when the mixture of PPD/H2O2/RES and PPD/H2O2 was applied to the RhE, some of the parameters such as morphological changes including the presence of apoptotic cells, barrier loss and increased IL-1 alpha release were observed. The results indicate that the mixture of ingredients used in permanent hair dyes have an irritant effect in RhE while the ingredients alone not.

The hair-dyeing ingredient, p-phenylenediamine (PPD), was previously reported to be mutagenic, possibly by inducing oxidative stress. However, the exact mechanism of PPD in inducing oxidative stress upon skin exposure during hair-dyeing in human keratinocytes remains unknown. The aim of our studies was therefore to investigate the toxicity of PPD and its by-products in human immortalized keratinocytes (HaCaT) after auto-oxidation and after reaction with hydrogen peroxide (H2O2). We found that the PPD half maximal effective cytotoxic concentration (EC50) to HaCaT is 39.37 and 35.63 μg/mL after 24 and 48 h, respectively, without addition of H2O2 to induce oxidation. When PPD (10 or 100 μg/mL) is combined with 10.5 μg/mL of H2O2, intracellular ROS production by HaCaT after 1 h was significantly increased and enhanced levels of DNA damage were observed after 4 h of exposure. After 24 h incubations, 20 μg/mL of PPD increased the level of DNA oxidation in HaCaT. Also, we found that the in vitro reaction between PPD and H2O2, even below the maximum allowance by cosmetic industries, released hydroxyl radicals which can damage DNA. Taken together, we conclude that PPD alone and when combined with H2O2 increases the formation of reactive oxygen species in human keratinocytes, leading to oxidative stress and subsequent DNA damage. These alterations suggest that the mechanism by which PPD exposure, alone or combined with H2O2, damages keratinocytes by the formation of the high reactive HO∙ radicals.

Currently, there is a strong global trend towards the development of in vitro models to replace the use of animals in safety evaluation tests. Reconstructed Human Epidermis (RHE) models have been employed as an alternative method to animal testing of skin corrosion and irritation potential of chemical compounds. However, the consequences of an absence of the dermal compartment in these models should be considered since the cross-talk between fibroblasts and keratinocytes is fundamental for promoting proper epidermal stratification, homeostasis, inflammatory response and wound healing. In this study, we compare in-house developed models of Reconstructed Human Epidermis (i.e. USP-RHE) and full thickness skin (i.e. USP-FTS) regarding their response when submitted to skin corrosion assays, based on Guideline 431 (OECD). The results show that both models correctly classified the four substances tested (2-phenylethyl bromide, benzylacetone, lactic acid, octanoic acid) as corrosive or non-corrosive. Furthermore, we have demonstrated higher cell viability of the USP-FTS model compared to the USP-RHE model, a sign of its improved barrier function, following the exposure to the substances test on the corrosion assay. This emphasizes the importance of employing in vitro models that are more physiologically relevant and that better mimic the in vivo situation for the toxicological screening of substances.

Melanoma is a highly invasive and metastatic cancer with high mortality rates and chemoresistance. Around 50% of melanomas are driven by activating mutations in BRAF that has led to the development of potent anti-BRAF inhibitors. However resistance to anti-BRAF therapy usually develops within a few months and consequently there is a need to identify alternative therapies that will bypass BRAF inhibitor resistance. The curcumin analogue DM-1 (sodium 4-[5-(4-hydroxy-3-methoxy-phenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate) has substantial anti-tumor activity in melanoma, but its mechanism of action remains unclear. Here we use a synthetic lethal genetic screen in Saccharomyces cerevisiae to identify 211 genes implicated in sensitivity to DM-1 toxicity. From these 211 genes, 74 had close human orthologues implicated in oxidative phosphorylation, insulin signaling and iron and RNA metabolism. Further analysis identified 7 target genes (ADK, ATP6V0B, PEMT, TOP1, ZFP36, ZFP36L1, ZFP36L2) with differential expression during melanoma progression implicated in regulation of tumor progression, cell differentiation, and epithelial-mesenchymal transition. Of these TOP1 and ADK were regulated by DM-1 in treatment-naive and vemurafenib-resistant melanoma cells respectively. These data reveal that the anticancer effect of curcumin analogues is likely to be mediated via multiple targets and identify several genes that represent candidates for combinatorial targeting in melanoma.

Carbaryl (1-naphthyl-methylcarbamate), a broad-spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, the main etiologic factor for skin carcinogenesis. We hypothesized that carbaryl exposure may increase deleterious effects of UV solar radiation on skin melanocytes. This study aimed to characterize human melanocytes after individual or combined exposure to carbaryl (100 μM) and solar radiation (375 mJ/cm2). In a microarray analysis, carbaryl, but not solar radiation, induced an oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of Microphtalmia-associated Transcription Factor (MITF), the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Carbaryl and solar radiation induced a gene response suggestive of DNA damage and cell cycle alteration. The expression of CDKN1A, BRCA1/2 and MDM2 genes was notably more intense in the combined treatment group, in a synergistic manner. Flow cytometry assays demonstrated S-phase cell cycle arrest, reduced apoptosis levels and faster induction of cyclobutane pyrimidine dimers (CPD) lesions in carbaryl treated groups. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation.

Studies about cervical carcinogenesis have demonstrated the increased expression of matrix-metalloproteinase (MMP) according to the grade of cervical intraepithelial lesions. Considering the importance of innovative techniques to introduce noninvasive and rapid diagnoses for patients, this study aimed to perform MMP-9 immunocytochemistry in cervical smears according to the cytopathological diagnoses, in order to monitor MMP activity in cervical smears. This cross-sectional study investigated the expression of MMP-9 in normal cervical smears, inflammatory cervical smears, squamous intraepithelial lesions, and cervical carcinoma. Cervical smears from 630 women were collected for cytopathological diagnoses and immunocytochemistry. Women with squamous intraepithelial lesions showed an increase in MMP-9 expression, with moderate to intense staining occurring with increasing cervical lesion grade. The prevalence of moderate to intense MMP-9 staining was 9% in normal cervical smears, 12% in cervical inflammation, 24% in low-grade squamous intraepithelial lesion (LSIL), 92% in high-grade squamous intraepithelial lesions (HSIL) and 100% in cervical carcinoma cases. In the specific case of LSIL, we found that association with MMP-9 is more evident when there is the simultaneous presence of an infectious agent. Thus, the expression of MMP-9 in cervical smears increases according to the grade of cervical lesion and LSIL in the presence of infectious agents showed higher MMP-9 expression than women with LSIL without infectious agents. Diagn. Cytopathol. 2014;42:827–833. © 2014 Wiley Periodicals, Inc.

Skin aging is a natural process of the human body that may be accelerated due to extrinsic causes. Libidibia ferrea, popularly known as juca, is a small tree, which possesses an abundant phenolic composition with potential antioxidant and enzymatic inhibition activities. Thus, this work aimed to investigate the anti-wrinkle and anti-whitening potentials of juca trunk bark (LFB) and pod (LFP) extracts. A comprehensive analysis of LFB and LFP phenolic composition was accomplished by means of liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Effects on skin degradation were assessed by inhibitory enzymatic activity against elastase, hyaluronidase and collagenase through colorimetric assays. Cellular viability in B16F10 and primary fibroblasts were determined by Trypan Blue exclusion assay. Anti-melanogenic effects on B16F10 cells were evaluated using cellular tyrosinase, melanin content, western blot, and RT-qPCR analyses. Inhibition of matrix metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) was determined by gelatin zymography and western blot methodologies. LC–MS/MS analyses of LFB and LFP extracts allowed the characterization of 18 compounds, among them, flavonoids, phenolic acids, and secoridoids. Additionally the pod and trunk bark compositions were compared. Hyaluronidase inhibitory activity for both extracts, LFB (IC50 = 8.5 ± 0.8 µg/mL) and LFP (IC50 = 16 ± 0.5 µg/mL), was stronger than standard rutin (IC50 = 27.6 ± 0.06). Pro-MMP-2 was significantly inhibited by both extracts. LFB and LFP decreased the melanin content in B16F10 due to tyrosinase inhibitory activity. L. ferrea extracts has high potential as a cosmetic ingredient due to its anti-wrinkle and depigmentant effects.

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases involved in the extracellular matrix degradation. MMP-2 and MMP–9 are overexpressed in several human cancer types, including melanoma, thus the development of new compounds to inhibit MMPs' activity is desirable. Molecular dynamic simulation and molecular properties calculations were performed on a set of novel β-N-biaryl ether sulfonamide-based hydroxamates, reported as MMP-2 and MMP-9 inhibitors, for providing data to develop an exploratory analysis. Thermodynamic, electronic, and steric descriptors have significantly discriminated highly active from moderately and less active inhibitors of MMP-2 whereas apparent partition coefficient at pH 1.5 was also significant for the MMP-9 data set. Compound 47 was considered an outlier in all analysis, indicating the presence of a bulky substituent group in R3 is crucial to this set of inhibitors for the establishment of molecular interactions with the S1 subsite of both enzymes, but there is a limit. © 2012 Wiley Periodicals, Inc.

Summary Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

Skin, the largest organ of the human body, is organized into an elaborate layered structure consisting mainly of the outermost epidermis and the underlying dermis. A subcutaneous adipose-storing hypodermis layer and various appendages such as hair follicles, sweat glands, sebaceous glands, nerves, lymphatics, and blood vessels are also present in the skin. These multiple components of the skin ensure survival by carrying out critical functions such as protection, thermoregulation, excretion, absorption, metabolic functions, sensation, evaporation management, and aesthetics. The study of how these biological functions are performed is critical to our understanding of basic skin biology such as regulation of pigmentation and wound repair. Impairment of any of these functions may lead to pathogenic alterations, including skin cancers. Therefore, the development of genetically controlled and well characterized skin models can have important implications, not only for scientists and physicians, but also for manufacturers, consumers, governing regulatory boards and animal welfare organizations. As cells making up human skin tissue grow within an organized three-dimensional (3D) matrix surrounded by neighboring cells, standard monolayer (2D) cell cultures do not recapitulate the physiological architecture of the skin. Several types of human skin recombinants, also called artificial skin, that provide this critical 3D structure have now been reconstructed in vitro. This review contemplates the use of these organotypic skin models in different applications, including substitutes to animal testing.

The antioxidant capacity of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazoles was evaluated. The values of antioxidant capacities of compounds 2d and 2e were found to be, respectively, 2,700 ± 150 and 3,135 ± 230 TE by the ORAC method, corresponding to a significant antioxidant capacity. recent publication, we reported the antioxidant capacity of phenylpyrazole using the ORAC method (14). In continua- tion of our research program, we report here the antioxidant evaluation of 2-(3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4- phenylthiazoles, synthetic derivatives compounds of facile preparation (Scheme 1).

Melanoma accounts for only 4% of skin malignancies, but it has a high mortality rate. Though the specific inhibitors of MAPK pathway (BRAFi and MEKi) have revolutionized melanoma treatment, most of patients are subject to resistance, justifying the constant search for new therapeutic compounds. Recent data from our laboratory indicated that 4-nerolidylcathecol (4-NC), extracted from Pothomorphe umbellata, a native Brazilian plant, induces apoptosis in melanoma cells by ROS production, DNA damage and p53 expression increase and promoted an antiproliferative effect in reconstructed skin. In this work, we aim to evaluate the 4-NC efficacy in overcoming resistance to BRAF and MEK inhibitors in melanoma cell lines as well in xenograft models. Firstly, the vemurafenib-resistant (R) and trametinib/vemurafenib-resistant (DR) melanoma cell lines were generated and characterized by MTT, fluorescence microscopy and western blotting. The cytotoxicity was evaluated by Trypan Blue Exclusion and Clonogenic Assays. The invasion potential was demonstrated by 2D and 3D models. Besides that, SK-MEL-103 melanoma cells (5x105 cells) were injected s.c. in female BALB/c nude mice. Once the tumors appeared, treatments were initiated by i.p. injection for 3 times a week for 3 weeks with 4-NC (10mg/kg), DMSO or no treatment. 4-NC showed cytotoxicity (IC50 ~30µM), colony formation and invasion decrease in vitro assays. To further investigate that, we dissected the molecular pathways involved with induction of Endoplasmic Reticulum (ER) Stress by Unfolded Protein Response (UPR). As results, we found many altered proteins in naïve, R and DR melanoma cells, especially regarding elevated levels of BiP and CHOP. The latter has been implicated in apoptotic events and was confirmed by Flow cytometry. 4-NC, previously reported as a proteasome inhibitor, was able to significantly suppress the xenograft tumor growth by 4-fold compared to controls, with complete tumor remission in one animal. P53, p16 and cleaved PARP expression were increased in the tumors of treated animals indicating apoptosis and cell cycle arrest. MMP2 and MMP14 gene expression were decreased in the same samples, demonstrating a promising role of 4-NC in the inhibition of melanoma invasion also in animal models. Although preliminary, our data suggest that 4-NC compound has an important cytotoxic effect in melanoma models and should be better investigated for future applications in human cutaneous melanoma treatment including resistant patients. Approval of Ethics Committee: Office CEUA/FCF/70-2012 - USP/Brazil. Financial support: FAPESP (2014/06959-0); CNPq (385282/2011-7). Citation Format: Debora Kristina Alves-Fernandes, Erica Aparecida de Oliveira, Fernanda Faião-Flores, Silvana Sandri, Walter Turato, Silvia Berlanga de Moraes Barros, Silvia Stuchi Maria-Engler. ER stress induced by 4-nerolidylcathecol (4-NC) promotes antitumor effects in BRAFi/MEKi sensitive and resistant melanoma models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A14.

BACKGROUND: There has been growing interest in sodium copper chlorophyllin (Cu-Chl) as a food colourant and supplement owing to its beneficial biological activities. Studies have revealed that this green pigment inhibits experimental carcinogenesis and interacts with proteins and genotoxic agents. Health-related activities have also been associated with the prevention of lipid peroxidation. However, intestinal absorption of this pigment has been considered insignificant, raising questions of whether eventual biological properties are related to pre- or post-absorptive actions. In this study, intestinal absorption of Cu-Chl and its appearance in serum and organs were estimated by high-performance liquid chromatography analysis in rat feeding experiments. The effect of ingested Cu-Chl on lipid peroxidation was analysed by measuring thiobarbituric acid-reactive substances and antioxidant enzyme activities in hepatic and brain tissues of oxidative stress-induced rats. RESULTS: The two main components of commercial Cu-Chl, namely Cu-chlorin e6 and Cu-chlorin e4, showed different digestive behaviours, and only Cu-chlorin e4 was found in serum, liver and kidneys. Antioxidant activity in vivo could be observed in brain and seemed to be related to in situ protection but not to antioxidant enzyme modulation. CONCLUSION: As at least one of the major components of Cu-Chl is effectively absorbed, further pharmacokinetic studies are encouraged to access absorption rates and the role of ingested copper chlorophyllins in mammals. Copyright © 2009 Society of Chemical Industry

Background: We aimed to evaluate the ascorbic acid concentration in secondary aqueous humour (AH) from glaucomatous patients and to compare it with primary AH from primary open-angle glaucoma patients and non-glaucomatous patients. Methods: Primary AH samples were prospectively obtained from clinically uncontrolled primary open-angle glaucoma patients and senile cataract patients (controls) prior to trabeculectomy and cataract surgery. Secondary AH samples were obtained from eyes with previous intraocular surgery, prior to trabeculectomy or cataract surgery. AH (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber just before surgery and then immediately stored at −80°C. The ascorbic acid concentration was determined in a masked fashion by high-pressure liquid chromatography. Results: A total of 18 patients with senile cataract, 16 glaucomatous patients with primary AH (no previous intraocular surgery) and 11 glaucomatous patients with secondary AH (previous intraocular surgery) were included. There was no difference in mean age between groups (P = 0.15). The mean ± standard deviation concentration of ascorbic acid in the secondary AH from glaucomatous patients (504 ± 213 µmol/L [95% confidence interval {CI}, 383–624]) was significantly lower than the concentration of ascorbic acid found in the primary aqueous of primary open-angle glaucoma (919 ± 427 µmol/L [95% CI, 709–1128]) and control patients (1049 ± 433 µmol/L [95% CI, 848–1249]; P

A maioria das pesquisas realizadas até hoje sobre os efeitos benéficos da pariparoba (Pothomorphe umbellata L. Miq) foi feita com o extrato da raíz desta espécie, e o seu emprego em larga escala comprometeria a exploração sustentável deste insumo natural. Neste sentido o uso das folhas da pariparoba, ao invés de raízes pela indústria cosmética, não põe em risco a existência da espécie. Neste trabalho foi determinada a concentração de 4-nerolidilcatecol (4-NC) de um extrato de folhas por metodologia analítica validada em nosso laboratório, cujo valor foi cerca de 30% menor que no extrato de raiz, obtido da mesma maneira. Na avaliação de fotoestabilidade do extrato de folhas, uma solução de 0,25 mg/mL não apresentou alterações significativas do perfil espectroscópico após 2 horas de exposição à radiação UVB, demonstrando sua estabilidade. Metaloproteinases (MMPs) são endopeptidases dependentes de zinco, envolvidas na remodelagem da matriz extracelular (MEC), e importantes na formação das rugas típicas do fotoenvelhecimento cutâneo. Também avaliamos, por técnica de zimografia, a capacidade do extrato de folhas de P. umbellata de inibir a atividade de MMPs 2 e 9 in vitro de pele de camundongos sem pêlo. O extrato de folhas (0,1 mg/mL) inibiu em 80% a atividade destas enzimas, conforme avaliação densitométrica. Most researches that have been done until today about the beneficial effects of pariparoba (Pothomorphe umbellata L. Miq) have been done with root extract of this species, but the use in large scale would compromise the sustainable exploration of this natural resource. In this sense, the utilization of pariparoba leaves, substituting the roots, in the cosmetic industry does not put in risk the existence of the species. In this work the concentration of 4-nerolidylcathecol (4-NC) in leaf extract was determined by the analytical methodology validated in our laboratory. The concentration of 4-NC in leaf extract was around 30% less than that of root extract, obtained in the same way. Concerning the study of the photostability of a leaves extract solution containing 4-NC did not demonstrate meaningful alterations in the spectrometry profile after 2 hours of exposure under UVB radiation, showing its stability under this conditions. Metalloproteinases (MMPs) are endopeptidases that are zinc-dependent, involved in remodeling extracellular matrix (ECM), that are important in the appearance of typical photoaging wrinkles. In this work the capacity of leaf extract of P. umbellata to inhibit MMP-2 and 9 activities of hairless mouse skin in vitro by zymography gel was also evaluated. The leaf extract (0,1 mg/mL) inhibit in 80% activity of this enzymes, according to the densitometric zymography evaluation.

A maioria das pesquisas realizadas até hoje sobre os efeitos benéficos da pariparoba (Pothomorphe umbellata L. Miq) foi feita com o extrato da raíz desta espécie, e o seu emprego em larga escala comprometeria a exploração sustentável deste insumo natural. Neste sentido o uso das folhas da pariparoba, ao invés de raízes pela indústria cosmética, não põe em risco a existência da espécie. Neste trabalho foi determinada a concentração de 4-nerolidilcatecol (4-NC) de um extrato de folhas por metodologia analítica validada em nosso laboratório, cujo valor foi cerca de 30% menor que no extrato de raiz, obtido da mesma maneira. Na avaliação de fotoestabilidade do extrato de folhas, uma solução de 0,25 mg/mL não apresentou alterações significativas do perfil espectroscópico após 2 horas de exposição à radiação UVB, demonstrando sua estabilidade. Metaloproteinases (MMPs) são endopeptidases dependentes de zinco, envolvidas na remodelagem da matriz extracelular (MEC), e importantes na formação das rugas típicas do fotoenvelhecimento cutâneo. Também avaliamos, por técnica de zimografia, a capacidade do extrato de folhas de P. umbellata de inibir a atividade de MMPs 2 e 9 in vitro de pele de camundongos sem pêlo. O extrato de folhas (0,1 mg/mL) inibiu em 80% a atividade destas enzimas, conforme avaliação densitométrica.Most researches that have been done until today about the beneficial effects of pariparoba (Pothomorphe umbellata L. Miq) have been done with root extract of this species, but the use in large scale would compromise the sustainable exploration of this natural resource. In this sense, the utilization of pariparoba leaves, substituting the roots, in the cosmetic industry does not put in risk the existence of the species. In this work the concentration of 4-nerolidylcathecol (4-NC) in leaf extract was determined by the analytical methodology validated in our laboratory. The concentration of 4-NC in leaf extract was around 30% less than that of root extract, obtained in the same way. Concerning the study of the photostability of a leaves extract solution containing 4-NC did not demonstrate meaningful alterations in the spectrometry profile after 2 hours of exposure under UVB radiation, showing its stability under this conditions. Metalloproteinases (MMPs) are endopeptidases that are zinc-dependent, involved in remodeling extracellular matrix (ECM), that are important in the appearance of typical photoaging wrinkles. In this work the capacity of leaf extract of P. umbellata to inhibit MMP-2 and 9 activities of hairless mouse skin in vitro by zymography gel was also evaluated. The leaf extract (0,1 mg/mL) inhibit in 80% activity of this enzymes, according to the densitometric zymography evaluation.

A new high performance liquid chromatographic method has been established for simultaneous determination of chlorogenic acid, caffeic acid and caffeine in hydroalcoholic and aqueous extracts of Ilex paraguariensis. Analytical curves showed good linear regression in the concentration ranges 0.49-7.8 µg/mL for chlorogenic acid, 0.25-3.9 µg/mL for caffeic acid and 0.244-7.8 µg/mL for caffeine. Reduction of the DPPH· radical was used to determine the antioxidant capacity of the extracts. Our method for the simultaneous determination of chlorogenic acid, caffeic acid and caffeine was highly sensitive, having lower detection and quantitation limits than other papers that used similar methodology.Um novo método de cromatografia líquida de alta eficiência foi desenvolvido para a determinação simultânea de ácido clorogênico, ácido caféico e cafeína no extrato hidroalcoólico e aquoso de Ilex paraguariensis. As curvas de calibração mostraram boa regressão linear nas faixas de concentração 0,49-7,8 µg/mL para o ácido clorogênico, 0,25-3,9 µg/mL para ácido caféico e 0,244-7,8 µg/mL para cafeína. A redução do radical DPPH· foi usada para determinar a capacidade antioxidante dos extratos. Nosso método para a determinação simultânea de ácido caféico, ácido clorogênico e cafeína foi altamente sensível, possuindo limites de detecção e de quantificação menores do que em outros trabalhos que empregaram metodologias semelhantes.

The advanced glycation end products (AGEs) of proteins are common factors in the pathophysiology of a number of disorders related to aging. The skin generation of AGEs occurs mainly through nonenzymatic glycation reactions of extracellular matrix (ECM) proteins in the dermis. The AGEs have been touted as one of the factors responsible for healing impairment and loss of elasticity of healing skin, affecting growth, differentiation, and cellular motility, as well as cytokines response, metalloproteinases expression, and vascular hemostasis. In this study, we generated an in vitro full-thickness reconstructed skin based on a glycated collagen matrix dermal compartment to evaluate the effects of glycation on dermal ECM and ultimately on the epidermis. Epidermal differentiation and stratification patterns and the glycation-induced ECM changes were evaluated by histology, immunohistochemistry, and mRNA levels. In this study, we reported for the first time that changes in the dermal matrix caused by collagen I in vitro glycation processes also affect the epidermal compartment. We demonstrated that glycation of collagen induces expression of carboxymethyllysine in dermal and epidermal compartments and, consequently, an aging phenotype consisting of poor stratification of epidermal layers and vacuolization of keratinocyte cytoplasm. Increased expression of cell-cell adhesion markers, such as desmoglein and E-cadherin in glycated skins, is observed in the stratum spinosum, as well as an increased compression of dermal collagen matrix. We also submitted our 3D model of reconstructed glycated skin to screening of anti-AGE molecules, such as aminoguanidine, which prevented the glycated morphological status. Controlled human studies investigating the effects of anti-AGE strategies against skin aging are largely missing. In this context, we proposed the use of skin equivalents as an efficient model to investigate cellular interactions and ECM changes in the aging skin, and to elucidate the role of anti-AGEs molecules in this process.

The purpose of this chapter is to create awareness of global regulatory approaches outside the United States and the EU. This will make the reader aware of regulatory approaches and Risk Assessment in other regions. Risk assessment is an emerging concept in many regions of the globe. Most countries still legislate based in chemical hazard than risk. The initiatives and regulatory approaches in these countries are explored based on the knowledge and detail available. The chapter covers these topics in a survey style approach. The reader is encouraged to visit the references for more detailed information.

Phacoemulsification has been successfully employed in humans and animals for lens extraction. This ultrasonic extracapsular surgical technique induces hydroxyl radical formation in the anterior chamber, which accumulates despite irrigation and aspiration. In this paper we determined the total antioxidant status of aqueous humor after phacoemulsification by measuring aqueous humor ascorbic acid levels. Mixed-breed dogs (n = 11; weighing about 10 kg) with normal eyes as determined by slit-lamp biomicroscopy, applanation tonometry, and indirect ophthalmoscopy had phacoemulsification performed in one eye with the other eye used as a control. Samples of aqueous humor were obtained by anterior chamber paracentesis before surgery and at days 1, 2, 3, 7, and 15 after surgery. Total aqueous humor antioxidant status was inferred from the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis (2-amidopropane) chlorine. Ascorbic acid concentrations were measured by high-pressure liquid chromatography with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by anova followed by the paired t-test. Total antioxidant capacity was reduced from 48 to 27 min during the first 24 h with a gradual increase thereafter, remaining statistically lower than the control eye until 7 days postoperatively. Reduced levels of ascorbic acid followed this reduction in antioxidant capacity (from 211 to 99 microm after 24 h), remaining lower than the control eye until 15 days postoperatively. Protein concentration in aqueous humor increased from 0.62 mg/mL to 30.8 mg/mL 24 h after surgery, remaining statistically lower than the control eye until 15 days postoperatively. Paracentesis alone did not significantly alter the parameters measured. These results indicate that after phacoemulsification, the aqueous humor ascorbic acid levels and antioxidant defenses in aqueous humor are reduced, indirectly corroborating free radical production in the anterior chamber as a result of phacoemulsification. The inflammatory process consequent to the surgical procedure demonstrated by increased protein content in aqueous humor can also contribute to free radical production and ascorbic acid consumption.

Due to its antioxidant and photoprotective properties, Pothomorphe umbellata is a promising candidate for use in cosmetic and pharmaceutical formulations. These properties arise from the presence of 4-nerolidylcathecol (4-NC), a polyphenolic compound isolated from P. umbellata roots. This study investigates its photostability properties, as well as the chemical and the in vitro sun protection factor (SPF) of P. umbellata root extract in a gel formulation. A high performance liquid chromatography method was used to evaluate the chemical stability using 4-NC as marker at 5, 25 and 45 degrees C for 103 days. The photostability and the sun protection factor were analyzed by ultraviolet (UV) spectophotometry using samples irradiated with UVB lamp. No significant difference of the 4-NC concentration was found in formulations stored at 5 and 25 degrees C. All samples stored at 45 degrees C, however, showed degradation of gel structure. After 2h of UVB exposure, there was no change in the absorption profile of 4-NC. The sun protection factor of P. umbellata root extract gel to final concentration of 0.1% 4-NC was not expressive (SPF=3.35+/-0.02), suggesting the predominance of its antioxidant activity.

Foi avaliada a influência dos fatores (1) tempo: 10 e 40 minutos; (2) tamanho de partícula: 840 e 420 mm; (3) hidromódulo: 1:50 e 1:100 e (4) temperatura: 40 e 60 °C na extração do 4-nerolidilcatecol, (4-NC) mediante planejamento fatorial "2(4)" em que quatro variáveis foram estudadas em dois níveis (máximo e mínimo). O método de extração foi maceração e a quantificação do 4-nerolidilcatecol foi realizada por cromatografia liquida de alta eficiência (CLAE) com detetor eletroquímico. Os resultados da análise fatorial indicam que o fator principal que favorece a extração do princípio ativo é o tamanho de partícula [efeito (2): 12,2086]. Diminuindo o tamanho de partícula aumenta três vezes a quantidade de 4-nerolidilcatecol extraída, enquanto o tempo de maceração [efeito (1): -0,64198], hidromódulo [efeito (3): 1,069804] e temperatura [efeito (4): -0,64198] não influenciam de forma significativa a extração. As interações de dois fatores: (2:3) tamanho:hidromódulo: 1,181142; (2:1) tamanho:tempo: 0,9435065 e (2:4) tamanho:temperatura: 0,0817575 mostraram que apesar do fator tamanho(2) ter favorecido o processo, quando combinado com os demais fatores, não aumenta a eficiência da extração. A metodologia de otimização mediante análise fatorial aplicada ao processo de extração do 4-nerolidilcatecol demonstra a importância do estudo das interações e não de cada fator isolado.The influence of factors: (1) time, 10 and 40 minutes, (2) particle size, 840 and 420 mm; (3) hydromodule, 1:50 and 1:100, and (4) temperature, 40 and 60 °C, in the extraction of 4-nerolidylcathecol (4-NC) from the roots of P. umbellata using factorial design "2(4)" was studied in two levels (maximal and minimal). The extraction method was maceration and the measurement of 4-NC was by HPLC with electrochemical detection. The results of the factorial analysis indicated that the main factor that increases the extraction of the active principle is particle size [effect (2): 12.2086]. The reduction of the particle size (mesh 60) increases threefold the amount of 4-nerolidylchatecol in the extract, while the time of maceration [effect (1): -0.64198], hydromodule [effect (3): 1.069804] and temperature [effect (4): -0.64198] practically do not influence the extraction. Interaction between two factors (2:3) size-hydromodule 1.181142, (2:1) size-time 0.9435065 and (2:4) size-temperature 0.0817575, showed that although the main factor size (2) increases the efficiency of the process, when one of the other three factors was taken together the amount of 4-NC extracted was not significantly increased. The technique of optimization using factorial analysis to investigate the extraction of 4-nerolylilchatecol showed to be useful to notice the interactions between factors and not only the effect of each isolated factor.

There is a high degree of concern regarding the secure use of plant extracts and, for this very reason, preclinical and clinic toxicological evaluation of these extracts are needed. With the aim to assure the quality and the safety of the extract and due to the scarcity of literature information about Pariparoba extract toxicity, our purpose was to investigate the acute and subchronic toxicity of the standardized ethanolic dried root extract of Pothomorphe umbellata L. Miq. This extract was administered orally to adult swiss mice and wistar rats and the mutagenic potencial of the extract was also evaluated. The extract showed to be non toxic.Existe uma grande preocupação quanto ao uso seguro de extratos vegetais e, por esta razão, a necessidade de estudos toxicológicos pré-clínicos e clínicos destes extratos. O objetivo deste trabalho foi o de avaliar a toxicidade aguda e subcrônica do extrato hidroalcoólico liofilizado de Pothomorphe umbellata L. Miq., administrado por via oral para animais de laboratório. O potencial mutagênico do extrato foi também avaliado pelo teste do micronúcleo. Os resultados dos estudos a curto e médio prazo demonstraram que o extrato não apresenta propriedades tóxicas.

The oral dosages formulations from Pothomorphe umbellata (Piperaceae) can only be therapeutically evaluated after establishing 4-nerolidylcatechol (4-NRC) pharmacokinetic profile in plasma. For that purpose, a unique analytical method validation using HPLC-UV detection was developed for analysis of rat plasma samples. The animals received 10 mg/kg b.w. of 4-NRC i.v.. Analytical conditions were set with C18 column, methanol: acetonitrile: water (62:20:18) as mobile phase, and a flow rate of 1.6 mL/min. The assay was linear from 1.0 - 80.0 mcg/mL. The recovery procedure was performed by liquid-liquid extraction showing quantitative extraction (106.4±8.7%). Limit of Quantification (LOQ) was found to be 1.0 mcg/mL. Interassay precision and accuracy ranged from values of 14.4-3.0% and 24.0-0.2%, respectively. Recovery after liquid-liquid extraction was found to be 106.5 ± 3%. The method is simple and reliable with total run time of less than 15 min.

This work evaluated the levels of 5-aminolevulinic acid (ALA) in the liver of rats exposed to different doses of HCB (25,50, and 100 mg/kg b.w. for 4 weeks) and correlated them with lipid peroxidation parameters. Levels of ALA were determined by high-pressure liquid chromatography after derivatization with acetylacetone and formaldehyde, followed by fluorescence detection. The methodology was carefully validated, nonetheless hepatic levels of ALA in all animals treated or not were below the detection limit of the method (2.27mg of ALA/g liver). On the other hand, lipid peroxidation, evaluated as thiobarbituric acid reactants production and chemiluminescence was found significantly increased in the livers of all treated rats, in comparison with control values (pEste trabalho avaliou os níveis de ácido delta-aminolevulínico (ALA) em fígado de ratos expostos a diferentes doses de hexaclorobenzeno (HCB) (25, 50 e 100 mg/kg de peso corpóreo) durante 4 semanas e correlacionou com os parâmetros de peroxidação lipídica. Os níveis de ALA foram determinados por cromatografia líquida de alta eficiência após derivatização com acetilacetona e formaldeído, seguida de detecção de fluorescência. A metodologia foi cuidadosamente validada, apesar disso, os níveis hepáticos de ALA em todos os animais tratados ou não foram abaixo do limite de detecção do método (2,27 mg de ALA/ g de fígado). Por outro lado, para a peroxidação lipídica, avaliada como produção de reagentes ao ácido tiobarbitúrico e quimiluminescência, os resultados foram significativamente elevados em todos os animais tratados em comparação com os do grupo controle (p

The scientific establishment has been discussing the relationship between aging and oxidative stress for quite some time now. While we are still far from a general agreement about this subject, there is an impressive amount of data collected that can be used to draw a compelling picture of the events that take place during the human aging process and their correlation with the oxidant status of the organism. In this review, we bring forth the results of some key studies that can help to elucidate the aging-oxidative stress puzzle, as well as to explain which are the fundamental events in this interplay and why their causal relationships remain so elusive. We also put forward here data on the systemic oxidative stress status of a group of 503 healthy human subjects. The data consist of the plasma levels of TBARS and of the nutritional antioxidants, alpha-tocopherol, beta-carotene and ascorbic acid, and of the activity of the antioxidant enzymes, Cu, Zn-superoxide dismutase, catalase and glutathione peroxidase, of red blood cells. The data indicate that a moderate situation of oxidative stress gradually develops during human aging.

We determined the antioxidant status of the aqueous humor after extracapsular lens extraction in 14 mongrel dogs weighing about 10 kg. The animals were examined by slit lamp biomicroscopy, applanation tonometry and indirect ophthalmoscopy. One eye was submitted to conventional extracapsular lens extraction and the other was used as control. Samples of aqueous humor were obtained by anterior chamber paracentesis before and at days 1, 2, 3, 7 and 15 after surgery. Total antioxidant status was determined as the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis(2-amidopropane) chlorine. Ascorbic acid concentration was measured by HPLC with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by ANOVA followed by the Tukey-Kramer test. Protein concentration increased from 0.61 to 22 mg/ml 24 h after surgery. These levels were maintained and returned to normal at day 7. Total antioxidant capacity was reduced from 50 to about 30 min until day 3 and at day 7 it was equal to control. Ascorbic acid levels were reduced from 252 to about 110 microM and then returned to control values at day 15. Considering the importance of ascorbic acid concentration in aqueous humor for the maintenance of the antioxidant status of the anterior segment of the eye, the decrease of antioxidant defenses suggests that the surgical procedures promote an oxidative stress condition in the eye.

O objetivo do nosso trabalho foi desenvolver um método para avaliação da concentração do ±-tocoferol, considerado o antioxidante lipofílico de maior importância, e do 4-nerolidilcatecol (4-NC), uma substância natural com comprovada ação antioxidante in vitro e in vivo, em matriz biológica (homogeneizado de pele). Utilizamos a cromatografia de alta eficiência acoplada a um detector eletroquímico, sendo que o método apresentou linearidade para as concentrações de 0,025 µg/mL a 0,1 µg/mL para o ±-T (tempo de retenção 3, 4 min) e de 0,15 µg/mL a 2,5 µg/mL para o 4-NC (tempo de retenção 2,06 min), dissolvidos em etanol e etanol:água (1:1). A taxa de recuperação do ±-T adicionado nas concentrações de 0,5; 0,1 e 0,025 µg/mL aos homogeneizados de pele foi de 94,03; 111,2 e 80,7%, respectivamente. A taxa de recuperação de 4-NC adicionado nas concentrações de 2,5; 0,625 e 0,156 µg/mL foi de 103,7; 91,7 e 91,7%. Este método analítico foi e está sendo empregado, com sucesso devido à sua precisão e rapidez, em diversas análises do laboratório.Topical administration of antioxidants, such as ±-tocopherol (±-T), provides an efficient manner of enriching the endogenous cutaneous protection system, and it constitutes a successful strategy for diminishing the ultraviolet radiation-mediated oxidative damage. Besides ±-tocopherol the use of other natural occurring compounds with antioxidant activity has been proposed for the same purpose. The aim of this study was to develop a validated analytical method for the determination of a-tocopherol and 4-nerolidylcathecol (4-NC) concentrations in skin homogenates in a pharmaceutical formulations. We employed liquid chromatography with electrochemical detection. Chromatography was performed on a Supelcosil LC-8, 3 mm, 75x4.6 mm column (Supelco, Bellefonte, PA, USA) with a mobile phase of methanol:water (9:1) for 4-NC and (95:5) for a-T, both containing 20 mM LiClO4 and 2 mM KCl. The flow rate was set at 1.0 ml/min. We established validation parameters including sensitivity, precision, accuracy, stability and found a linear relationship between the concentrations ranges of 0.025 µg/mL to 0.1 µg/mL of ±-T and 0.15 mg/mL to 2.5 mg/mL of 4-NC. The recovery of ±-T from skin homogenates was 94.03, 111.2 and 80.7% for the concentrations of 0.5, 0.1 and 0.025 µg/mL respectively. The recovery for the following concentrations of 4-NC: 2.5, 0.625 and 0.156 µg/mL was 103.7, 91.7 and 91.7%. This analytical procedure has been successfully employed in cutaneous permeation studies, antioxidant activity studies and determinations of 4-NC in Pothomorphe umbellata root extracts.

Antioxidants such as vitamins E and C are known to play a significant role in ameliorating or preventing oxidative damage to the skin. However, to provide a satisfactory protection they must first permeate the skin, which serves as a permeation barrier. In this study we evaluated the influence of three different formulations (gel, gel–emulsion and emulsion) on the percutaneous absorption of 4-nerolidylcathecol, an antioxidant compound isolated from Pothomorphe umbellata root extracts. Also, the absorption of the isolated 4-nerolidylcathecol was compared with its absorption when dried P. umbellata root ethanolic extract was incorporated into a gel formulation. The ‘lag time method’ was employed for the analysis of the in vitro permeation data. All formulations showed satisfactory percutaneous penetration with the 4-nerolidylcathecol–gel presenting a higher rate of penetration leading to higher dry drug levels in the tissue.

Matrix metalloproteinases (MMP) are a group of enzymes related to extracelular matrix remodeling. Some types of MMP are overexpressed by malignant tumors, mainly the MMP-2 subtype, and have been associated to cancer invasiveness and metastasis. A receptor-independ- ent (RI) 4D-QSAR formalism was applied, herein, to a set of forty b-N-biaryl ether sulfonamide hydroxamates, previously reported as potent MMP-2 inhibitors, in order to map 3D- pharmacophore models and predict novel antitumor agents. The best RI 4D-QSAR model was statistically signifi- cant (N = 30, r 2 = 0.93, q 2 = 0.88, five occupancy descriptors (GCOD), LSE = 0.04, LOF = 0.11, outliers = 0), robust and not obtained by chance. The external predictability was 75 % (test set; N = 8). A different orientation (binding mode) in the MMP-2 catalytic site was suggested regarding the most hydrophobic portion (R1) of the compounds' structure. Compounds were predicted and their inhibitory activity against MMP-2 was calculated by using the optimum RI 4D-QSAR model. The findings have provided interesting in- formation to drive the designing and synthesis of novel po- tentially MMP-2 inhibitors against melanoma invasion.