Cytotoxic and genotoxic effects of two hair dyes used in the formulation of black color

According to the International Agency for Research on Cancer (IARC), some hair dyes are considered mutagenic and carcinogenic in in vitro assays and exposed human populations. Epidemiological studies indicate that hairdressers occupationally exposed to hair dyes have a higher risk of developing bladder cancer. In Brazil, 26% of the adults use hair dye. In this study, we investigated the toxic effects of two hair dyes, Basic Red 51 (BR51) and Basic Brown 17 (BB17), which are temporary dyes of the azo group (R-N=N-R'), used in the composition of the black hair dye. To this end, MTT and trypan blue assays (cytotoxicity), comet and micronucleus assay (genotoxicity) were applied, with HepG2 cells. For cytotoxic assessment, dyes were tested in serial dilutions, being the highest concentrations those used in the commercial formula for hair dyes. For genotoxic assessment concentrations were selected according to cell viability. Results showed that both dyes induced significant cytotoxic and genotoxic effects in the cells, in concentrations much lower than those used in the commercial formula. Genotoxic effects could be related to the azo structure present in the composition of the dyes, which is known as mutagenic and carcinogenic. These results point to the hazard of the hair dye exposure to human health.

Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri

Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the safe and compatible integrated use of biopesticides.

The triad indole-3-acetic acid ethyl ester/esterase/horseradish peroxidase as a new cytotoxic prodrug/enzyme combination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antioxidant, anti-acetylcholinesterase and cytotoxic activities of ethanol extracts of peel, pulp and seeds of exotic Brazilian fruits Antioxidant, anti-acetylcholinesterase and cytotoxic activities in fruits

Ethanol extracts of powdered genipap (Genipa americana L), umbu (Spondia tuberosa A.) and siriguela (Spondia purpurea L) prepared from separate pulp, seeds and peel were investigated for their (i) antioxidant capacity, which was evaluated by various known methods; (ii) acetylcholinesterase (AChE) inhibitory activity; and (iii) cytotoxic effect on corneal epithelial cells of sheep. The highest values of total phenolic content were obtained with peel and seed extracts. Siriguela and umbu (seeds and peel) extracts displayed the highest antioxidant activities. Lipid peroxidation assays using mimetic biomembranes and mouse liver homogenates indicated that genipap pulp is a promising antioxidant. The investigation of phenols and organic acid contents revealed the presence of quercetin, citric and quinic acids, chlorogenic acid derivatives, among others, in several extracts, with the highest amount found in siriguela seeds. Genipap pulp and siriguela seed ethanol extracts presented an AChE inhibition zone similar to that of the positive control, carbachol. AChE inhibition assay with chlorogenic acid, one of the main constituents of siriguela seeds, revealed that this acid showed activity similar to that of the control physostigmine. These data suggest that these extracts are potentially important antioxidant supplements for the everyday human diet, pharmaceutical and cosmetic industries. (C) 2012 Elsevier Ltd. All rights reserved.

Dillapiole as Antileishmanial Agent: Discovery, Cytotoxic Activity and Preliminary SAR Studies of Dillapiole Analogues

In this paper, the isolation of dillapiole (1) from Piper aduncum was reported as well as the semi-synthesis of two phenylpropanoid derivatives [di-hydrodillapiole (2), isodillapiole (3)], via reduction and isomerization reactions. Also, the compounds' molecular properties (structural, electronic, hydrophobic, and steric) were calculated and investigated to establish some preliminary structureactivity relationships (SAR). Compounds were evaluated for in vitro antileishmanial activity and cytotoxic effects on fibroblast cells. Compound 1 presented inhibitory activity against Leishmania amazonensis (IC50?=?69.3 mu M) and Leishmania brasiliensis (IC50?=?59.4 mu M) and induced cytotoxic effects on fibroblast cells mainly in high concentrations. Compounds 2 (IC50?=?99.9 mu M for L. amazonensis and IC50?=?90.5 mu M for L. braziliensis) and 3 (IC50?=?122.9 mu M for L. amazonensis and IC50?=?109.8 mu M for L. brasiliensis) were less active than dillapiole (1). Regarding the molecular properties, the conformational arrangement of the side chain, electronic features, and the hydrophilic/hydrophobic balance seem to be relevant for explaining the antileishmanial activity of dillapiole and its analogues.

2-Acetylpyridine- and 2-benzoylpyridine-derived hydrazones and their gallium(III) complexes are highly cytotoxic to glioma cells

2-Acetylpyridine-phenylhydrazone (H2AcPh), its para-chlorophenylhydrazone (H2AcpClPh) and para-nitrophenylhydrazone (H2AcpNO(2)Ph) analogues, the corresponding 2-benzoylpyridine-derived hydrazones (H2BzPh, H2BzpClPh and H2BzpNO(2)Ph) and their gallium(III) complexes were assayed for their cytotoxic activity against U87 (expressing wild-type p53 protein) and T98 (expressing mutant p53 protein) glioma cells. IC50 values against both glioma cells and against the MRC5 (human fetal lung fibroblast) lineage were obtained for the hydrazones, but not for their gallium(III) complexes, due to their low solubility. Hydrazones were highly cytotoxic at nanomolar doses against U87 and T98 cells. The therapeutic indexes (TI = IC50MRC5/IC50glioma) were 2-660 for T98 cells and 28-5000 for U87 cells, indicating that the studied hydrazones could be good antitumor drug candidates to treat brain tumors. (C) 2012 Elsevier Masson SAS. All rights reserved.

Evaluation of cytotoxic, genotoxic and antigenotoxic potential of Solanum lycocarpum fruits glicoalkaloid extract in V79 cells

Solanum lycocarpum St.-Hil (Solanaceae) is a hairy shrub or small much-branched tree of the Brazilian Cerrado, popularly known as "fruit-of-wolf". Considering that the induction of chromosomal mutations is involved in the process of carcinogenesis, and that S. lycocatpum is often used in folk medicine, it becomes relevant to study its effect on genetic material. In this sense, the aim of present study was to determine the possible cytotoxic, genotoxic and antigenotoxic potentials of S. lycocarpum fruits glycoalkaloid extract (SL) in Chinese hamster lung fibroblasts (V79 cells). The cytotoxicity was evaluated by the colony forming assay, apoptosis and necrosis assay. Trypan blue exclusion dye method and mitotic index. Genotoxic and antigenotoxic potential were evaluated by comet and chromosomal aberrations assays. Four concentrations of SL (4, 8, 16 and 32 mu g/mL) were used for the evaluation of its genotoxic potential. The DNA damage-inducing agent methyl methanesulfonate (MMS, 221 mu g/mL) was utilized in combination with extract to evaluate a possible protective effect. The results showed that SL was cytotoxic at concentrations above 32 mu g/mL by the colony forming assay. For apoptosis and necrosis assay, the concentration of 64 mu g/mL of SL showed statistically significant increase in cell death by apoptosis and necrosis, while the concentrations of 128 and 256 mu g/mL of SL demonstrated statistically significant increase in cell death by necrosis, compared with the control group. Analysis of cell viability by Trypan blue exclusion indicated >96% viability for treatments with concentrations up to 32 mu g/mL of SL No significant differences in MI were observed between cultures treated with different concentrations of 51 (4, 8, 16 and 32 mu g/mL) alone or in combination with MMS and the negative control, indicating that these treatments were not cytotoxic. The comet and chromosomal aberrations assays revealed that SL does not display genotoxic activity. Moreover, the different concentrations of SL showed protective effect against both genomic and chromosomal damages induced by MMS. (C) 2012 Elsevier Ltd. All rights reserved.

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-L-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-L-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human. (C) 2012 Published by Elsevier Masson SAS.

In vivo assessment of specific cytotoxic T lymphocyte killing

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells

In vitro generation and characterization of acute myeloid leukemia-reactive CD8 + cytotoxic T-lymphocyte clones from healthy donors

Donor-derived CD8+ cytotoxic T lymphocytes (CTLs) eliminating host leukemic cells mediate curative graft-versus-leukemia (GVL) reactions after allogeneic hematopoietic stem cell transplantation (HSCT). The leukemia-reactive CTLs recognize hematopoiesis-restricted or broadly expressed minor histocompatibility and leukemia-associated peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on recipient cells. The development of allogeneic CTL therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of current techniques for generating leukemia-reactive CTLs from unprimed healthy donors in vitro. In this work, a novel allogeneic mini-mixed lymphocyte/leukemia culture (mini-MLLC) approach was established by stimulating CD8+ T cells isolated from peripheral blood of healthy donors at comparably low numbers (i.e. 10e4/well) with HLA class I-matched primary AML blasts in 96-well microtiter plates. Before culture, CD8+ T cells were immunomagnetically separated into CD62L(high)+ and CD62L(low)+/neg subsets enriched for naive/central memory and effector memory cells, respectively. The application of 96-well microtiter plates aimed at creating multiple different responder-stimulator cell compositions in order to provide for the growth of leukemia-reactive CTLs optimized culture conditions by chance. The culture medium was supplemented with interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-2. In eight different related and unrelated donor/AML pairs with complete HLA class I match, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines of donor and patient origin nor primary B cell leukemias expressing the corresponding HLA restriction element. CTLs expressed T cell receptors of single V-beta chain families, indicating their clonality. The vast majority of CTL clones were obtained from mini-MLLCs initiated with CD8+ CD62L(high)+ cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10e8-10e10 cells within six to eight weeks. The capability of mini-MLLC derived AML-reactive CTL clones to inhibit the engraftment of human primary AML blasts was investigated in the immunodeficient nonobese diabetic/severe combined immune deficient IL-2 receptor common γ-chain deficient (NOD/SCID IL2Rγnull) mouse model. The leukemic engraftment in NOD/SCID IL2Rγnull was specifically prevented if inoculated AML blasts had been pre-incubated in vitro with AML-reactive CTLs, but not with anti-melanoma control CTLs. These results demonstrate that myeloid leukemia-specific CTL clones capable of preventing AML engraftment in mice can be rapidly isolated from CD8+ CD62L(high)+ T cells of healthy donors in vitro. The efficient generation and expansion of these CTLs by the newly established mini-MLLC approach opens the door for several potential applications. First, CTLs can be used within T cell-driven antigen identification strategies to extend the panel of molecularly defined AML antigens that are recognizable by T cells of healthy donors. Second, because these CTLs can be isolated from the stem cell donor by mini-MLLC prior to transplantation, they could be infused into AML patients as a part of the stem cell allograft, or early after transplantation when the leukemia burden is low. The capability of these T cells to expand and function in vivo might require the simultaneous administration of AML-reactive CD4+ T cells generated by a similar in vitro strategy or, less complex, the co-transfer of CD8-depleted donor lymphocytes. To prepare clinical testing, the mini-MLLC approach should now be translated into a protocol that is compatible with good manufacturing practice guidelines.

In vitro detection of cytotoxic T and NK cells in peripheral blood of patients with various drug-induced skin diseases

BACKGROUND: Cytotoxic cells are involved in most forms of drug-induced skin diseases. Till now, no in vitro test addressed this aspect of drug-allergic responses. Our report evaluates whether drug-induced cytotoxic cells can be detected in peripheral blood of nonacute patients with different forms of drug hypersensitivity, and also whether in vitro detection of these cells could be helpful in drug-allergy diagnosis. METHODS: GranzymeB enzyme-linked immunosorbent spot-forming (ELISPOT) and cell surface expression of the degranulation marker CD107a were evaluated on peripheral blood mononuclear cells from 12 drug-allergic patients in remission state and 16 drug-exposed healthy controls. RESULTS: In 10/12 allergic patients culprit but not irrelevant drug elicited granzymeB release after 48-72 h stimulation. It was clearly positive in patients with high proliferative response to the drug, measured in lymphocyte transformation tests. In patients, who showed moderate or low proliferation and low drug-response in granzymeB ELISPOT, overnight preincubation with interleukin (IL)-7/IL-15 enhanced drug-specific granzymeB release and allowed to clearly identify the offending agent. CD107a staining was positive on CD4+/CD3+, CD8+/CD3+ T cells as well as CD56+/CD3- natural killer cells. None of the drug-exposed healthy donors reacted to the tested drugs and allergic patients reacted only to the offending, but not to tolerated drugs. CONCLUSION: GranzymeB ELISPOT is a highly specific in vitro method to detect drug-reacting cytotoxic cells in peripheral blood of drug-allergic patients even several years after disease manifestation. Together with IL-7/IL-15 preincubation, it may be helpful in indentifying the offending drug even in some patients with weak proliferative drug-response.

Quantum dot cytotoxicity in vitro: An investigation into the cytotoxic effects of a series of different surface chemistries and their core/shell materials

Abstract The aim of this study was to assess the effects of a series of different surface coated quantum dots (QDs) (organic, carboxylated [COOH] and amino [NH(2)] polytethylene glycol [PEG]) on J774.A1 macrophage cell viability and to further determine which part of the QDs cause such toxicity. Cytotoxic examination (MTT assay and LDH release) showed organic QDs to induce significant cytotoxicity up to 48 h, even at a low particle concentration (20 nM), whilst both COOH and NH(2) (PEG) QDs caused reduced cell viability and cell membrane permeability after 24 and 48 h exposure at 80 nM. Subsequent analysis of the elements that constitute the QD core, core/shell and (organic QD) surface coating showed that the surface coating drives QD toxicity. Elemental analysis (ICP-AES) after 48 h, however, also observed a release of Cd from organic QDs. In conclusion, both the specific surface coating and core material can have a significant impact on QD toxicity.

Cytotoxic steroidal saponins from the rhizomes of Tacca integrifolia

Three new steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (1), (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (3), and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (5), as well as the new pregnane glycoside (3beta,16beta)-3-{[6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranosyl]oxy}-20-oxopregn-5-en-16-yl (4R)-5-(beta-D-glucopyranosyloxy)-4-methylpentanoate (6), were isolated from the rhizomes of Tacca integrifolia together with two known (25R) configurated steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (2) and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (4). The cytotoxic activity of the isolated compounds was evaluated in HeLa cells and showed the highest cytotoxicity value for compound 2 with an IC(50) of 1.2+/-0.4 muM. Intriguingly, while compounds 1-5 exhibited similar cytotoxic properties between 1.2+/-0.4 (2) and 4.0+/-0.6 muM (5), only compound 2 showed a significant microtubule-stabilizing activity in vitro.

Cytotoxic effects of polycarbonate-based orthodontic brackets by activation of mitochondrial apoptotic mechanisms

OBJECTIVES: The aim of the study was to evaluate the biological effects of water eluents from polycarbonate based esthetic orthodontic brackets. METHODS: The composite polycarbonate brackets tested were Silkon Plus (SL, fiber-glass-reinforced), Elan ME (EL, ceramic particle-reinforced) and Elegance (EG, fiber-glass-reinforced). An unfilled polyoxymethylene bracket (Brilliant, BR) was used as control. The brackets' composition was analyzed by ATR-FTIR spectrometry. The cytotoxicity and estrogenicity of the eluents obtained after 3months storage of the brackets in water (37°C) were investigated in murine fibroblasts (NIH 3T3), breast (MCF-7) and cervical cancer (CCl-2/Hela) cell lines. RESULTS: SL and EG were based on aromatic-polycarbonate matrix, whereas EL consisted of an aromatic polycarbonate-polyethylene terepthalate copolymer. A significant induction of cell death and a concurrent decrease in cell proliferation was noted in the EG eluent-treated cells. Moreover, EG eluent significantly reduced the levels of the estrogen signaling associated gene pS2, specifically in MCF7 cells, suggesting that cell death induced by this material is associated with downregulation of estrogen signaling pathways. Even though oxidative stress mechanisms were equally activated by all eluents, the EG eluents induced expression of apoptosis inducing factor (AIF) and reduced Bcl-xL protein levels. SIGNIFICANCE: Some polycarbonate-based composite brackets when exposed to water release substances than activate mitochondrial apoptosis.

Synthesis and cytotoxic activity of non-naturally substituted 4-oxycoumarin derivatives

Coumarins are a large family of natural and synthetic compounds exerting different pharmacological effects, including cytotoxic, anti-inflammatory or antimicrobial. In the present communication we report the synthesis of a series of 12 diversely substituted 4-oxycoumarin derivatives including methoxy substituted 4-hydroxycoumarins, methyl, methoxy or unsubstituted 3-aryl-4-hydroxycoumarins and 4-benzyloxycoumarins and their anti-proliferative effects on breast adenocarcinoma cells (MCF-7), human promyelocytic leukemia cells (HL-60), human histiocytic lymphoma cells (U937) and mouse neuroblastoma cells (Neuro2a). The most potent bioactive molecule was the 4-hydroxy-5,7-dimethoxycoumarin (compound 1) which showed similar potency (IC(50) 0.2-2 μM) in all cancer cell lines tested. This non-natural product reveals a simple bioactive scaffold which may be exploited in further studies.