Toxicology in Vitro (TOXICOL IN VITRO)

Publications in this journal

• Article: The force of the spontaneously contracting zebrafish heart, in the assessment of cardiovascular toxicity: application on adriamycin

  Triantafyllia Orfanidou, Chrisovalantis Papaefthimiou, Efthimia Antonopoulou, Ioannis Leonardos, George Theophilidis
  Toxicology in Vitro 01/2013;
• Article: Heme oxygenase-1 mediates the protective role of quercetin against ethanol-induced rat hepatocytes oxidative damage

  Liu S, Hou W, Yao P, Li N, Zhang B, Hao L, Nüssler AK, Liu L
  Toxicology in Vitro 02/2012; 26(1):74.
• Article: Quercetin protects against ethanol-induced oxidative damage in rat primary hepatocytes

  Liu S, Hou W, Yao P, Zhang B, Sun S, Nüssler AK, Liu L
  Toxicology in Vitro 03/2010; 24(2):516.
• Article: Micromolar Zn(2+) potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: possible disturbance of intracellular Ca(2+) and Zn(2+) homeostasis.

  Akio Kinazaki, Yoko Sakanashi, Tomohiro M Oyama, Haruka Shibagaki, Kohei Yamashita, Erika Hashimoto, Yumiko Nishimura, Shiro Ishida, Yoshiro Okano, Yasuo Oyama
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  ABSTRACT: Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn(2+) at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn(2+) permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn(2+) at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn(2+), we have cytometrically examined the effects of econazole, ZnCl(2), and their combination on rat thymocytes. ZnCl(2) at concentrations ranging from 1 microM to 30 microM significantly increased the lethality induced by 10 microM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 microM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl(2), suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl(2). Econazole at 0.3 microM or more in the presence of ZnCl(2) increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca(2+). Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+), was also augmented by econazole at 0.1 microM or more in the presence of ZnCl(2). Results suggest that the combination of submicromolar econazole with micromolar ZnCl(2) may increase the intracellular concentration of Ca(2+) and Zn(2+), leading to disturbance of intracellular Ca(2+) and Zn(2+) homeostasis that triggers cytotoxic action.
  Toxicology in Vitro 07/2009; 23(4):610-6.
• Article: In vitro absorption of metal powders through intact and damaged human skin.

  Francesca Larese Filon, Flavia D'Agostin, Matteo Crosera, Gianpiero Adami, Massimo Bovenzi, Giovanni Maina
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  ABSTRACT: The bioavailability of metals, which are known as important contact allergens, is decisive for the development and the maintenance of contact dermatitis. The aim of this study was to evaluate the percutaneous penetration of metal powders of cobalt (Co), nickel (Ni) and chromium (Cr) and the effect of skin lesions on skin absorption. In vitro permeation experiments were performed using the Franz diffusion cells with intact and damaged human skin. Physiological solution was used as receiving phase and metal powders (Co, Ni and Cr) dispersed in synthetic sweat at pH 4.5 were applied as donor phase to the outer surface of the skin for 24h. The amount of each metal permeating the skin was analysed by electro-thermal atomic absorption spectroscopy (ETAAS). Donor solution analysis demonstrated that metals were present as ions. Measurements of metals skin content were also exploited. Median Co and Ni concentrations found in the receiving phase were significantly higher when Co and Ni powders were applied on the abraded skin than after application on the intact skin (3566 and 2631ngcm(-2) vs. 8.4 and 31ngcm(-2), respectively). No significant difference was found in Cr permeation through intact and damaged skin. The measurement of metals skin content showed that Co, Ni and Cr concentrations were significantly higher in the damaged skin than in the intact skin. Co and Ni ions concentrations increased significantly when the donor solutions were applied on the damaged skin, while Cr ions concentrations did not increase. This study demonstrated that Co and Ni powders can permeate through damaged skin more easily than Cr powder, which has probably a stronger skin proteins binding capacity. Therefore, our results suggest that is necessary to prevent skin contamination when using toxic substances because a small injury to the skin barrier can significantly increase skin absorption.
  Toxicology in Vitro 07/2009; 23(4):574-9.
• Article: Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells.

  Abdolamir Allameh, Shahnaz Esmaeli, Somaieh Kazemnejad, Masoud Soleimani
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  ABSTRACT: The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.
  Toxicology in Vitro 07/2009; 23(4):674-9.
• Article: In vitro eye irritancy test of lauryl derivatives using the reconstructed rabbit corneal epithelium model.

  Sanae Matsuda, Masayoshi Hisama, Hiroharu Shibayama, Norihiko Itou, Masahiro Iwaki
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  ABSTRACT: The rabbit corneal epithelium model (RCE model) was developed as a three-dimensional in vitro model to replace animal testing for the assessment of eye tolerance. In the model, a stratified culture of rabbit corneal epithelial cells is grown at the air-liquid interface on an amniotic membrane acting as a parabasal membrane. The alkaline exposure was restored each day in the presence of no irritants, although with the addition of SLS, which is a major irritant, the restoration of deficit was inhibited on the RCE model in a dose-dependent manner. The results of this test were comparable with those of the Draize test, and thus, this method using the RCE model may prove to be a useful and sensitive in vitro eye irritation test. The lauryl fatty chain derivatives, such as polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-l-glutaminate (SLG), which are widely used as surfactants for toiletry products and cosmetics, were evaluated for in vitro eye irritation potential using the RCE model. SLS, PLE, SPLE, MGL, and SLG inhibited 88.7%, 59.2%, 69.0%, 47.5%, and 15.7% of the restoration of deletion 24h after treatment at a concentration of 0.05%. The IC(50) (50% inhibitory concentration) values of SLS, PLE, SPLE, MGL, and SLG were 0.002%, 0.021%, 0.005%, 0.056%, and 0.448%, respectively. These results indicated that a functional group at the end of lauryl chain is an important factor for inhibiting the restoration of deletion using the RCE model.
  Toxicology in Vitro 07/2009; 23(4):555-60.
• Article: Evaluation of changes of cell-surface thiols as a new biomarker for in vitro sensitization test.

  Mie Suzuki, Morihiko Hirota, Shigenobu Hagino, Hiroshi Itagaki, Setsuya Aiba
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  ABSTRACT: In order to find a novel biomarker for a simple assay to predict skin sensitization, we evaluated cell-surface thiols as a biomarker reflecting intracellular signaling in THP-1 cells (human monocytic cell line). First, we found that a decrease of cell-surface thiols on hapten-treated THP-1 cells was induced in parallel with phosphorylation of p38 MAPK. Next, we confirmed that 2-mercaptoethanol in the culture medium and the differentiation state of THP-1 cells did not affect the changes of cell-surface thiols by hapten. Changes of cell-surface thiols on THP-1 cells were detected after 2h treatment with most allergens (e.g., DNCB, NiSO(4)), as well as some non-allergens (e.g., Tween80, benzalkonium chloride), though other non-allergens (e.g., SDS, glycerol) had no effect. When either a significant decrease or increase of cell-surface thiols (more than 15% in each case) was used as a criterion, the results using 36 allergens and 16 non-allergens were in good accordance with those of in vivo assays. Finally, we confirmed that ATP, which is released as a consequence of cytotoxicity, did not affect the changes of cell-surface thiols. Our results suggest that changes of cell-surface thiols may be useful for an in vitro sensitization assay, which we designate as the SH test.
  Toxicology in Vitro 07/2009; 23(4):687-96.
• Article: Evaluation of antigenotoxic activity of isoliquiritin apioside from Glycyrrhiza glabra L.

  Prabhjit Kaur, Satwinderjeet Kaur, Neeraj Kumar, Bikram Singh, Subodh Kumar
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  ABSTRACT: Prevention of manifestation of events characteristic of carcinogenesis is being emphasized a rational strategy to combat cancer. Reactive oxygen species (ROS) play an important role in tumor initiation through oxidative damage of DNA. In search for lead molecules in cancer chemoprevention from natural products, a fraction 'Rlicca' isolated from Glycyrrhiza glabra was studied for modulatory effect against hydrogen peroxide and 4-nitroquinoline-N-oxide induced genotoxicity in Escherichiacoli PQ37 using SOS chromotest and in human peripheral blood lymphocytes using the Comet assay. The fraction 'Rlicca' at a concentration of 191 microM decreased the SOS inducing potency (SOSIP) of hydrogen peroxide (1.0mM) and NQO (20 microg/ml) by 83.72% and 68.77%, respectively. In the human blood lymphocytes, 'Rlicca' reduced the tail moment induced by hydrogen peroxide (25 microM) and NQO (5 microg/ml) by 88.04% and 76.64%, respectively, using the Comet assay. The spectroscopic data of 'Rlicca' fraction revealed it to be isoliquiritin apioside, a chalcone oligoglycoside. This is the first report of isoliquiritin apioside with marked potential to combat oxidative stress-induced genotoxicity.
  Toxicology in Vitro 07/2009; 23(4):680-6.
• Article: Estrogenic activity of cadmium, copper and zinc in the yeast estrogen screen.

  Xavier Denier, Elisabeth M Hill, Jeanette Rotchell, Christophe Minier
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  ABSTRACT: Heavy metals are increasingly studied due to their apparent ability to disrupt signaling pathways of living organisms including humans. Among various mechanisms of action, metals are suspected of exerting estrogenic activity in human and wildlife. In this study, a wide range of concentration of cadmium, copper, lead, mercury and zinc (from 95.4 pM to 1 mM) alone or in combination with the natural estrogen, 17-beta estradiol, has been tested using the yeast estrogen screen, an estrogen receptor dependent transcriptional expression assay. No direct trans-activation of the estrogen-responsive element could be measured with any of the concentration of the metals tested. Nevertheless, cadmium, copper and zinc were able to potentiate the estradiol-induced response in a dose-dependent manner. Significant stimulation was obtained from 10 nM cadmium, 100 nM copper and 2 nM zinc. Maximum response led to decrease of the estradiol EC50 by a factor 10. This study indicates that cadmium, copper and zinc can act as potential endocrine disrupters by modulating the estrogenic activity of endogenous hormones.
  Toxicology in Vitro 07/2009; 23(4):569-73.
• Article: Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2.

  Cyril Vidau, Jean-Luc Brunet, Alexandra Badiou, Luc P Belzunces
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  ABSTRACT: Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.
  Toxicology in Vitro 07/2009; 23(4):589-97.
• Article: Cytotoxic activity of sanguinarine and dihydrosanguinarine in human promyelocytic leukemia HL-60 cells.

  Jirí Vrba, Petr Dolezel, Jaroslav Vicar, Jitka Ulrichová
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  ABSTRACT: The benzo[c]phenanthridine alkaloid sanguinarine has been studied for its antiproliferative activity in many cell types. Almost nothing however, is known about the cytotoxic effects of dihydrosanguinarine, a metabolite of sanguinarine. We compared the cytotoxicity of sanguinarine and dihydrosanguinarine in human leukemia HL-60 cells. Sanguinarine produced a dose-dependent decline in cell viability with IC(50) (inhibitor concentration required for 50% inhibition of cell viability) of 0.9 microM as determined by MTT assay after 4h exposure. Dihydrosanguinarine showed much less cytotoxicity than sanguinarine: at the highest concentration tested (20 microM) and 24h exposure, dihydrosanguinarine decreased viability only to 52%. Cytotoxic effects of both alkaloids were accompanied by activation of the intrinsic apoptotic pathway since we observed the dissipation of mitochondrial membrane potential, induction of caspase-9 and -3 activities, the appearance of sub-G(1) DNA and loss of plasma membrane asymmetry. This aside, sanguinarine also increased the activity of caspase-8. As shown by flow cytometry using annexin V/propidium iodide staining, 0.5 microM sanguinarine induced apoptosis while 1-4 microM sanguinarine caused necrotic cell death. In contrast, dihydrosanguinarine at concentrations from 5 microM induced primarily necrosis, whereas apoptosis occurred at 10 microM and above. We conclude that both alkaloids may cause, depending on the alkaloid concentration, both necrosis and apoptosis of HL-60 cells.
  Toxicology in Vitro 06/2009; 23(4):580-8.
• Article: Exposure of the murine RAW 264.7 macrophage cell line to hydroxyapatite dispersions of various composition and morphology: assessment of cytotoxicity, activation and stress response.

  Julia Scheel, Sabine Weimans, Astrid Thiemann, Eckhard Heisler, Martina Hermann
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  ABSTRACT: Cellular stress responses leading to the release of cytotoxic mediators are discussed as indicators of the hazard presented by particles, and in particular ultrafine particles or nanomaterials. The present study was designed to investigate effects of the following materials on RAW 264.7 macrophages: three hydroxyapatite materials of various morphologies, i.e., nano-sized with rod-like (HA-NR), plate-like (HA-NP) or needle-shaped (HA-NN) morphology, and an irregularly shaped composite of hydroxyapatite and protein (HPC) in the low micrometer range. Concentrations of 50, 100, 500, 1000 and 5000 microg/ml were applied and cells were analyzed for viability (XTT-test), cytokine production (TNF-alpha) and induction of nitric oxide (NO) after 18 and 42 h. DQ12 quartz and lipopolysaccharide (LPS) served as positive controls. Up to concentrations of 500 microg/ml, cell viability was not considerably impaired by the test samples at both timepoints. Overall, viability was about one order of magnitude higher than with comparable concentrations of quartz. TNF-alpha release was induced in all samples after 18 h, with HA-NR showing the most pronounced induction at 100 microg/ml, still clearly below the LPS signal. No or little induction was observed after 42 h. NO production was low after 18 and 42 h. The results support the conclusion that the tested materials exhibit good biocompatibility and are safe to use.
  Toxicology in Vitro 05/2009; 23(3):531-8.
• Article: In vitro cytotoxic and immunomodulatory profiling of low molecular weight polyethylenimines for pulmonary application.

  Andrea Beyerle, Sabrina Höbel, Frank Czubayko, Holger Schulz, Thomas Kissel, Achim Aigner, Tobias Stoeger
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  ABSTRACT: Polyethylenimines (PEI) are potent non-viral nucleic acid delivery vehicles used for gene delivery and RNA interference (RNAi). For non-invasive pulmonary RNAi therapy the respiratory tissue is an attractive application route, but offers particularly unwanted side-effects like cytotoxicity as well as inflammatory and immune responses. In the current study, we determined the most crucial issues of pulmonary applications for two low molecular weight PEIs in comparison to the well-known lung toxic crystalline silica. Cytotoxic effects and inflammatory responses were evaluated in three murine pulmonary target cell lines, the alveolar epithelial (LA4), the alveolar macrophage (MH-S) and the macrophage-monocyte-like (RAW 264.7) cell line. For both PEIs, cytotoxicity was detected most prominently in the alveolar epithelial cells and only at high doses. Cytokine responses, in contrast were observed already at low PEI concentrations and could be divided into three groups, induced (i) by free PEI (IL-6, TNF-alpha, G-CSF), (ii) by PEI/siRNA complexes (CCL2, -5, CXCL1, -10), or (iii) unaffected by either treatment (IL-2, -4,-7, -9, and CCL3). We conclude that even for the respiratory tissue both PEIs represent powerful siRNA delivery tools with reduced cytotoxicity and minor proinflammatory potency. However, in relation to response levels observed upon crystalline silica exposures, some PEI induced proapoptotic and proinflammatory responses might not be considered completely harmless, therefore further in vivo investigations are advisable.
  Toxicology in Vitro 05/2009; 23(3):500-8.
• Article: In vitro selection and efficacy of topical skin protectants against the nerve agent VX.

  J Millerioux, C Cruz, A Bazire, G Lallement, L Lefeuvre, D Josse
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  ABSTRACT: Against highly toxic chemicals that are quickly absorbed in the skin, topical formulations could adequately complement specific protective suits and equipments. In this work, we evaluated in vitro and compared the skin protection efficacy against the nerve agent VX of four different topical formulations: oil-in-water and water-in-oil emulsions, a perfluorinated-based cream and a hydrogel. Semi-permeable silicone membrane, pig-ear and human abdominal split-thickness skin samples mounted in diffusion cells were compared as in vitro permeation tests. The results showed that silicone membrane could be used instead of skin samples to screen for potentially effective formulations. However, the results indicated that due to potentially significant interactions between formulations and skin, relevant ranking of formulations according to their protective efficacy could require tests with skin samples. The main phase of emulsions, water or oil, was not found to be critical for skin protective efficacy against VX. Instead, specific film-forming ingredients such as perfluorinated-based polymers and silicones could significantly affect the skin protective efficacy of formulations. We showed that a hydrogel containing specific hydrophilic polymers was by far the most effective of the formulations evaluated against VX skin permeation in vitro.
  Toxicology in Vitro 05/2009; 23(3):539-45.
• Article: Aflatoxin B1 misregulates the activity of serine proteases: possible implications in the toxicity of some mycotoxin.

  M Cuccioloni, M Mozzicafreddo, S Barocci, F Ciuti, L Re, A M Eleuteri, M Angeletti
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  ABSTRACT: Aflatoxins are highly hazardous contaminants of common food and feed. Aflatoxin B1 in particular, the most predominant among aflatoxins, was thoroughly demonstrated to be highly toxic, mutagenic, teratogenic and carcinogenic in many animal species. Besides its established targets and effects, this work investigates on the possible direct interaction between aflatoxin B1 and three major serine proteases, namely elastase, thrombin and trypsin. These proteases belongs to a class of structurally and functionally related proteins pivotal in both direct and indirect regulation of a number of cellular events. Additionally, several pathological processes, including cancer, inflammatory processes and thrombosis, rely upon the subtle equilibrium between these enzymes and their potential modulators: in fact, their misregulation, caused by foreign molecules, could facilitate (or be the cause for) the occurrence of these pathologies. Our results provide the evidence for a reversible binding between AFB1 and these enzymes, likely to have profound implications in the manifestation of aflatoxicosis. Precisely, the toxin behaved as a moderate competitive inhibitor toward the enzymatic activity of the serine proteases in the low micromolar range.
  Toxicology in Vitro 05/2009; 23(3):393-9.