Description

Toxicology in Vitro will publish original research papers and occasional reviews on the use of in vitro techniques for determining the toxic effects of chemicals and elucidating their mechanisms of action. The Journal will encourage the submission of studies which, by utilising cell or tissue culture, perfused organs, tissue slices, isolated cells or subcellular fractions, including enzymes and cell-receptors, investigate the mechanisms of toxic effects encountered in vivo or better characterize the relationship between in vitro and in vivo observations. All aspects of toxicology will be covered, including specific organ toxicity (eg neurotoxicity, nephrotoxicity), various toxic phenomena such as carcinogenesis or teratogenesis, and the development, characterization and validation of new in vitro models for the assessment and study of toxicity. The Journal's editorial policy will be firmly rooted in the need for high-quality science in support of health or safety decisions, and emphasis will be placed on results that facilitate evaluation of the hazard of chemicals to man and animals. NEW! Special rate for ESTIV members now available. For more information contact Dr. Diane Benford, ESTIV Secretary, Robens Institute, University of Surrey, Guildford, Surrey, GU2 5XH, U.K.; Tel: +44 1483 259 204; Fax: +44 1483 503 517; E-mail: d.benford@surrey.ac.uk or visit the ESTIV website at http://leden.tref.nl/ruttend/

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Elsevier

Publications in this journal

• Human colon cell culture models of different transformation stages to assess conjugated linoleic acid and conjugated linolenic acid metabolism: challenges and chances.

  Authors: Christian Degen, Nina Habermann, Stefanie Piegholdt, Michael Glei, Gerhard Jahreis

  Toxicology in vitro : an international journal published in association with BIBRA.

  Both cellular transformation status and cell culture conditions affect fatty acid metabolism. Hence, the incorporation and metabolism of c9,t11-CLA (conjugated linoleic acid) and other CFAsBoth cellular transformation status and cell culture conditions affect fatty acid metabolism. Hence, the incorporation and metabolism of c9,t11-CLA (conjugated linoleic acid) and other CFAs (conjugated fatty acids) were compared in colon cells (LT-97, adenoma; HT-29, adenocarcinoma). Growth inhibition by CFA in LT-97 cells was assessed via the DAPI(4´,6-diamidino-2-phenylindole dihydrochloride) assay. Basal gene expression of desaturases (Δ5, Δ6 and Δ9) and elongases (1, 2, 5 and 6) was determined in LT-97 using PCR. Analysis of cellular fatty acids revealed a 2-fold higher incorporation of c9,t11-CLA (40μM and 80μM) in HT-29 cells compared to LT-97 cells. The β-oxidized and elongated conjugated dienoic (CD) fatty acids differed by 8-fold (CD-C16:2/CD-C20:2; HT-29: 8:1; LT-97: 1:1). Notably, LT-97 cells were shown to convert conjugated linolenic acid (CLnA) to CLA. Moreover, LT-97 cells revealed no basal expression of elongase 2. CLnA caused stronger growth inhibition (⩽80μM) compared to CLA (200μM). The results indicate that LT-97 cells represent a superior model to carry out elongation and desaturation studies of unsaturated and conjugated fatty acids compared to HT-29 cells. Nevertheless, further in-depth metabolic and transcriptomic analyses are required to confirm this suggestion.

• Polychlorinated Biphenyl Quinone Metabolites Lead to Oxidative Stress in HepG2 Cells and the Protective Role of Dihydrolipoic Acid.

  Authors: Jing Liu, Erqun Song, Lichao Liu, Xiaoyan Ma, Xingguo Tian, Hui Dong, Yang Song

  Toxicology in vitro : an international journal published in association with BIBRA.

  Parent polychlorinated biphenyls (PCBs) have been shown to induce cellular oxidative stress. However, the effects of PCB active metabolites have not been extensively investigated. Parent PCBs areParent polychlorinated biphenyls (PCBs) have been shown to induce cellular oxidative stress. However, the effects of PCB active metabolites have not been extensively investigated. Parent PCBs are first converted to hydroquinone metabolites via cytochrome P-450-catalyzed hydroxylation, and the hydroquinone metabolites are then further oxidized into the corresponding quinone metabolites. Quinones are responsible for a wide range of toxic effects because of their high reactivity. Previous studies have suggested that reactive oxygen species (ROS) play important roles in multiple toxic mechanisms. In this context, the present study was undertaken to investigate oxidative stress resulting from treatment with PCB quinones in HepG2 cells. The protective effects resulting from co-administration of dihydrolipoic Acid (DH-LA) were also investigated. We have found that exposure to PCB quinones leads to: (1) a decrease in cell viability; (2) an increase in both the total ROS production and superoxide production; (3) only 3Cl-PCBQ caused significant increase in the thiobarbituric acid reactive substances (TBARS) level; (4) an increase in SOD activity and a decrease in catalase activity; and (5) a decrease in GST activity and GSH level. We have also found that quinones possessing a higher number of chlorine atoms on the quinone ring display a greater activity and that DH-LA is an effective protective agent as it diminishes PCB quinone-induced cellular oxidative stress.

• Citrinin reduces testosterone secretion by inducing apoptosis in rat Leydig cells.

  Authors: Shuqiang Liu, Dan Wang, Junwen Zhang, Dongzhi Zhang, Meng Gong, Chao Wang, Ning Wei, Wenhua Liu, Yongqi Wang, Chongxue Zhao, Yaxiong Cui, Defu Hu

  Toxicology in vitro : an international journal published in association with BIBRA.

  A previous study has shown that CTN (Ctrinin) inhibits mouse testosterone production. In this study, the mechanism by which testosterone production is inhibited by CTN in rat Leydig cells wasA previous study has shown that CTN (Ctrinin) inhibits mouse testosterone production. In this study, the mechanism by which testosterone production is inhibited by CTN in rat Leydig cells was investigated, and the morphological evidence of apoptosis, including nuclei fragmentation and phosphatidylserine (PS) exposure on cell surfaces, was clearly observed 36 h after CTN exposure. The results showed that citrinin at 50 and 100 μM significantly suppressed testosterone secretion by human chorionic gonadotropin (hCG) at 10 IU/ml. Western blotting results showed that CTN induced formation of processed p53, caspase-9, and caspase-3 proteins in a dose-dependent manner; CTN also induced a dose-dependent increase in caspase-3 catalytic activity. Western blot assays also showed that CTN decreased expression of three key enzymes (P450scc, 3β-HSD-1, and StAR) of testosterone production. Taken together, these results suggested that CTN reduced testosterone secretion by inducing apoptosis in rat Leydig cells, a mechanism that might account for CTN stimulation of p53 expression followed by activation of multiple caspases.

• Critical roles of Rho-associated kinase in membrane blebbing and mitochondrial pathway of apoptosis caused by 1-butanol.

  Authors: Kanako Noritake, Toshihiko Aki, Takeshi Funakoshi, Kana Unuma, Akina Nara, Chizuru Kato, Koichi Uemura

  Toxicology in vitro : an international journal published in association with BIBRA.

  Alcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlyingAlcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlying the cytotoxicity of industrial alcohols, with the notable exception of ethanol. The goal of our current study is to elucidate the molecular mechanism of cytotoxicity caused by primary alcohols containing longer carbon chains than ethanol. We find that 1-butanol induces morphological changes in H9c2 cardiomyoblastoma including nuclear condensation and membrane blebbing, both of which are features of apoptotic response. Moreover, a decrease in the mitochondrial membrane potential, the cytosolic release of cytochrome C, and the activation of caspase9 and caspase3 was observed, thus revealing the activation of the mitochondrial apoptotic pathway by 1-butanol. The addition of Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed the membrane blebbing and mitochondrial apoptotic pathway. In comparison z-VAD-fmk, a pan-caspase inhibitor, did not inhibit membrane blebbing but did prevent cell death following exposure to 1-butanol. These results indicate that mitochondrial pathway of apoptosis and membrane blebbing are parallel phenomena that occur downstream of ROCK. This kinase thus plays an essential role in 1-butanol cytotoxicity and subsequent cell death in H9c2 cells.

• Automated neurosphere sorting and plating by the COPAS large particle sorter is a suitable method for high-throughput 3D in vitro applications.

  Authors: K Gassmann, J Baumann, S Giersiefer, J Schuwald, T Schreiber, H F Merk, E Fritsche

  Toxicology in vitro : an international journal published in association with BIBRA.

  Existing guidelines for testing developmental neurotoxicity (DNT) propose investigations in rodents, which are ethically questionable as well as time and cost intensive. Thus, there is internationalExisting guidelines for testing developmental neurotoxicity (DNT) propose investigations in rodents, which are ethically questionable as well as time and cost intensive. Thus, there is international agreement that predictive in vitro methods are needed to increase efficiency of testing and limit the number of animals used. One of a variety of novel approaches for DNT testing utilizes neurospheres, three-dimensional aggregate cultures of primary normal neural progenitor cells (NPCs). Because sorting and plating of single neurospheres is one of the most time-consuming steps within the assay, the aim of this study was to evaluate if the complex object parametric analyzer and sorter (COPAS PLUS(TM), Union Biometrica Inc.) is a suitable tool for automated sorting and plating of neurospheres. The results of the comparison of NPC viability, proliferation, migration, differentiation and intracellular oxidative stress between manually and COPAS sorted and plated neurospheres of different species show that the automation by the COPAS instrument does not influence the basic performance of neurospheres. Therefore, we consider the COPAS instrument as a useful tool for higher throughput neurosphere research in toxicology, neuroregeneration, brain development, drug development and brain aging research.

• Pre-synaptic function explains age-dependent actions of general anesthetics in the rat hippocampal synaptic transmission.

  Authors: Koki Hirota, Rika Sasaki, Mitsuaki Yamazaki

  Toxicology in vitro : an international journal published in association with BIBRA.

  Mechanisms by which age modifies general anesthetic requirements remain uncertain. In order to examine the age-related modification of general anesthetics in the central nervous system, we haveMechanisms by which age modifies general anesthetic requirements remain uncertain. In order to examine the age-related modification of general anesthetics in the central nervous system, we have studied the effects of thiopental and sevoflurane on hippocampal synaptic transmission in young and elderly rats. Field potentials of area CA1 were electrically elicited in hippocampal slices from young (4-month) and elderly (2-year) male Wistat rats. The effects of sevoflurane on both excitatory and inhibitory synaptic transmission were similar in the young and elderly preparations. In contrast, thiopental produced a greater effect on inhibitory synaptic transmission in young than elderly hippocampi, whereas the actions on excitatory synaptic transmission were negligible in both preparations. Corresponding experiments revealed (a) that the duration of recurrent inhibition was more prolonged by thiopental in young compared to elderly animals and (b) that thiopental enhanced the γ-amino-butyric acid (GABA) release from pre-synaptic terminals in an age-dependent manner. The thiopental actions on GABA discharge from pre-synaptic terminals appear to be responsible for the observed difference between young and elderly animals. The age-dependent reduction in neurotransmitter stores in pre-synaptic terminals may explain the age-related alterations in general anesthetic actions.

• Investigation of 2,6-diisopropylphenol (propofol)-evoked Ca(2+) movement and cell death in human glioblastoma cells.

  Authors: Wei-Zhe Liang, Chung-Ren Jan, Cheng-Hsien Lu

  Toxicology in vitro : an international journal published in association with BIBRA.

  This study examined whether propofol altered [Ca(2+)](i) and caused cell death in DBTRG-05MG cells. Propofol at 400-1000μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal wasThis study examined whether propofol altered [Ca(2+)](i) and caused cell death in DBTRG-05MG cells. Propofol at 400-1000μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was decreased partially by removal of extracellular Ca(2+). Propofol-induced Ca(2+) influx was not altered by nifedipine, econazole, SK&F96365, and protein kinase C (PKC) activators; but was inhibited by PKC inhibitor. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished propofol-induced [Ca(2+)](i) rise. Incubation with propofol inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished propofol-induced [Ca(2+)](i) rise. At 300-700μM, propofol killed cells in a concentration-dependent manner. The cytotoxic effect of propofol was partly reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining further showed that 300-500μM propofol evoked apoptosis. Propofol also increased reactive oxygen species (ROS) production. Overall, propofol induced a [Ca(2+)](i) rise by inducing PLC- and PKC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via non store-operated Ca(2+) channels. Propofol induced cell death that might involve ROS-mediated apoptosis.

• Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests.

  Authors: Jessica Lundqvist, Johanna El Andaloussi-Lilja, Christina Svensson, Helena Gustafsson Dorfh, Anna Forsby

  Toxicology in vitro : an international journal published in association with BIBRA.

  Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalized C17.2 cell line,Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalized C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3(rd) or 4(th) day to fresh N2 medium with supplements. After 7 days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.

• Paeonol from Hippocampus kuda Bleeler suppressed the neuro-inflammatory responses in vitro via NF-κB and MAPK signaling pathways.

  Authors: S W A Himaya, Bomi Ryu, Zhong-Ji Qian, Se-Kwon Kim

  Toxicology in vitro : an international journal published in association with BIBRA.

  Inflammation has recently been implicated as a critical mechanism responsible for neurodegenerative diseases. In this study, paeonol (1-(2-hydroxy-4-methoxyphenyl) ethanone) isolated from the seaInflammation has recently been implicated as a critical mechanism responsible for neurodegenerative diseases. In this study, paeonol (1-(2-hydroxy-4-methoxyphenyl) ethanone) isolated from the sea horse Hippocampus kuda Bleeler was studied as an agent to suppress LPS induced activation of BV-2 microglial and RAW264.7 macrophage cells. The results obtained showed that paeonol significantly suppressed LPS induced release of pro-inflammatory products such as nitric oxide (NO), prostaglandin E2 (PGE(2)), and cytokines; tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Furthermore, the compound down regulated the protein and gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β and IL-6 in both cell lines. Molecular signaling pathway studies showed that paeonol inhibited the translocation of nuclear factor-κB (NF-κB) p65 and p50 subunits to the nucleus by blocking IKKα/β (IκB kinase α/β) mediated degradation of IκBα. Moreover, it suppressed the phosphorylation of mitogen activated protein kinase (MAPK) pathway molecules; c-Jun N-terminal kinases (JNK) and p38 in both cell lines. Collectively these results indicate that paeonol blocked the LPS stimulated inflammatory responses in BV-2 and RAW264.7 cells via modulating MAPK and NF-κB signaling pathways. Therefore, paeonol could be a promising candidate to be used in neuro-inflammatory therapy.

• In vitro skin corrosion: human skin model test - a validation study.

  Authors: Gajanan Rajpal Deshmukh, Kuntrapakam Hema Kumar, Poojari Venkata Suresh Reddy, Boddapati Srinivasa Rao

  Toxicology in vitro : an international journal published in association with BIBRA.

  AbstractThe present article is an attempt to validate the in vitro skin corrosion human skin model test as per the OECD test guidelines 431 as an alternative method for in vivo skinAbstractThe present article is an attempt to validate the in vitro skin corrosion human skin model test as per the OECD test guidelines 431 as an alternative method for in vivo skin corrosion/irritation test. All over India, in vivo skin corrosion/irritation test is commonly used, rather than in vitro skin corrosion models. Hence, the present study was under taken with EpiSkinTM, SNC, Lyon, France to validate this in vitro model. Various corrosive and non corrosive chemicals with their known skin corrosive property were used to validate the study as specified under the guideline (OECD 431). The results obtained in this study were in accordance with the corrosive properties of the respective chemicals.

• Effects of cerium oxide nanoparticles to fish and mammalian cell lines: An assessment of cytotoxicity and methodology.

  Authors: P Rosenkranz, M L Fernández-Cruz, E Conde, M B Ramírez-Fernández, J C Flores, M Fernández, J M Navas

  Toxicology in vitro : an international journal published in association with BIBRA.

  Two cerium oxide nanoparticles (CeO(2) NPs) and one micro-sized CeO(2) particle were thoroughly characterized in their pristine form, in water and in cell culture medium. The particles were testedTwo cerium oxide nanoparticles (CeO(2) NPs) and one micro-sized CeO(2) particle were thoroughly characterized in their pristine form, in water and in cell culture medium. The particles were tested for cytotoxicity to the H4IIE rat hepatoma cell line or the RTG-2 rainbow trout gonadal cell line by means of four standard cytotoxicity assays. Nominal concentrations were verified by inductively coupled plasma mass spectrometry (ICP-MS) and methods were assessed for their suitability to detect reliably adverse effects due to particle exposure. All three particles showed aggregation in water and media. In the H4IIE cell line, the MTT cytotoxicity test revealed that negative effects could be observed for the CeO(2) NPs after 24h and for all particles after 72h of exposure, making the effects size, concentration and time dependent. No negative effect for the concentrations tested was detected for the remaining three assays and the RTG-2 cell line, making the MTT assay and the H4IIE cell line an appropriate system to assess adverse effects of CeO(2) NPs. A verification of the nominal concentration through ICP-MS revealed that there was a discrepancy between nominal and measured concentration depending on concentration and particle tested. Interferences of particles with assays were found to be present and need to be taken into consideration.

• Low levels of residual oil fly ash (ROFA) impair innate immune response against environmental mycobacteria infection in vitro.

  Authors: Verónica C Delfosse, Andrea K Gioffré, Deborah R Tasat

  Toxicology in vitro : an international journal published in association with BIBRA.

  Epidemiological studies have shown that pollution derived from industrial and vehicular transportation provokes adverse health effects causing broad spectrum of ambient respiratory diseases.Epidemiological studies have shown that pollution derived from industrial and vehicular transportation provokes adverse health effects causing broad spectrum of ambient respiratory diseases. Therefore, air pollution should be taken into account when microbial diseases are evaluated. Environmental mycobacteria (EM) are opportunist pathogens in a variety of immunocompromised patients eliciting significant impact on human morbidity and mortality. The aim of this study was to evaluate the in vitro effects of residual oil fly ash (ROFA) on the alveolar macrophages (AMs) response to opportunistic bacteria. AMs from young Wistar rats were obtained by bronchoalveolar lavage and co-cultured with Mycobacterium phlei (MOI 10). We exposed AM cultures to ROFA to characterize the effect of low ROFA concentrations (0, 2.5, and 5μg/ml) and evaluated the response of pre-exposed AM against the bacilli. Low ROFA concentrations induced superoxide anion and nitrites production (p<0.001). Pre-exposure to ROFA (2.5 and 5μg/ml) caused a significant reduction on TNFα (p<0.001) and superoxide anion (p<0.001) production but, did not modify the nitrite production when AM were co-cultured with M. phlei. In addition, ROFA significantly diminished AM killing ability in culture (p<0.001). Hence, our results indicate that pre-exposure to low levels of ROFA modifies the innate pulmonary defence mechanisms against environmental mycobacteria.

• A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts.

  Authors: Claudia N Martini, Matías Gabrielli, María Del C Vila

  Toxicology in vitro : an international journal published in association with BIBRA.

  Glyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not onlyGlyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not only glyphosate itself but also surfactants that may also be toxic. 3T3-L1 fibroblasts are a useful tool to study adipocyte differentiation, this cell line can be induced to differentiate by addition of a differentiation mixture containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine. We used this cell line to investigate the effect of a commercial formulation of glyphosate (GF) on proliferation, survival and differentiation. It was found that treatment of exponentially growing cells with GF for 48h inhibited proliferation in a dose-dependent manner. In addition, treatment with GF dilution 1:2000 during 24 or 48h inhibited proliferation and increased cell death, as evaluated by trypan blue-exclusion, in a time-dependent manner. We showed that treatment of 3T3-L1 fibroblasts with GF increased caspase-3 like activity and annexin-V positive cells as evaluated by flow cytometric analysis, which are both indicative of induction of apoptosis. It was also found that after the removal of GF, remaining cells were able to restore proliferation. On the other hand, GF treatment severely inhibited the differentiation of 3T3-L1 fibroblasts to adipocytes. According to our results, a glyphosate-based herbicide inhibits proliferation and differentiation in this mammalian cell line and induces apoptosis suggesting GF-mediated cellular damage. Thus, GF is a potential risk factor for human health and the environment.

• Cell death induced by the Alternaria mycotoxin Alternariol.

  Authors: Fatma Bensassi, Cindy Gallerne, Ossama Sharaf El Dein, Mohamed Rabeh Hajlaoui, Hassen Bacha, Christophe Lemaire

  Toxicology in vitro : an international journal published in association with BIBRA.

  Mycotoxins are unavoidable contaminants of most foods and feeds, and some are known to be detrimental to human health. It is thus worthwhile to understand how cells of the intestinal system, one ofMycotoxins are unavoidable contaminants of most foods and feeds, and some are known to be detrimental to human health. It is thus worthwhile to understand how cells of the intestinal system, one of the primary targets of these toxins, respond to their toxic effects. In this study, human colon carcinoma cells were used to elucidate the cell death mode and the pathways triggered by Alternariol (AOH), the most important mycotoxin produced by Alternaria species, which are the most common mycoflora infecting small grain cereals worldwide. Treatment of cells with AOH resulted in a loss of cell viability by inducing apoptosis. AOH-induced apoptosis was mediated through a mitochondria-dependent pathway, characterized by a p53 activation, an opening of the mitochondrial permeability transition pore (PTP), a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O(2)(-) and caspase 9 and 3 activation. Besides, deficiency of the pro-apoptotic protein Bax partially protected cells against AOH-induced mitochondrial alterations. In addition, experiments performed on purified mitochondria indicated that AOH does not directly target this organelle to induce cell death. Our results demonstrate for the first time that AOH-induced cytotoxicity is mediated by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells.

• Isatin-3-N(4)-benzilthiosemicarbazone, a non-toxic thiosemicarbazone derivative, protects and reactivates rat and human cholinesterases inhibited by methamidophos in vitro and in silico.

  Authors: Rômulo Pillon Barcelos, Rafael de Lima Portella, Thiago Henrique Lugokenski, Edovando José Flores da Rosa, Guilherme Pires Amaral, Luiz Filipe Machado Garcia, Leandro Bresolin, Vanessa Carratu, Félix Alexandre Antunes Soares, Nilda Berenice de Vargas Barbosa

  Toxicology in vitro : an international journal published in association with BIBRA.

  Organophosphates (OPs), which are widely used as pesticides, are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The inactivation of AChE results in the accumulation ofOrganophosphates (OPs), which are widely used as pesticides, are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The inactivation of AChE results in the accumulation of acetylcholine at cholinergic receptor sites, causing a cholinergic crisis that can lead to death. The classical treatment for OP poisoning is administration of oximes, but these compounds are ineffective in some cases. Here we determined whether the new compound isatin-3-N(4)-benzilthiosemicarbazone (IBTC), which in our previous study proved to be an antioxidant and antiatherogenic molecule, could protect and reactivate AChE and BChE. Toxicity of IBTC after subcutaneous injection in mice was measured using assays for oxidized diclorofluoresceine (DCF), thiobarbituric acid reactive substances (TBARS), non-protein thiol (NPSH) levels, and catalase (CAT), sodium potassium (Na(+)/K(+)) ATPase, delta-aminolevulinic acid dehydratase (ALA-D), and glutathione peroxidases (GPx) enzyme activities. The cytotoxicity was evaluated and the enzymatic activity of cholinesterase was measured in human blood samples. Molecular docking was used to predict the mechanism of IBTC interactions with the AChE active site. We found that IBTC did not increase the amount of DCF-RS or TBARS, did not reduce NPSH levels, and did not increase CAT, (Na(+)/K(+)) ATPase, ALA-D, or GPx activities. IBTC protected and reactivated both AChE and BChE activities. Molecular docking predicted that IBTC is positioned at the peripheral anionic site and in the acyl binding pocket of AChE and can interact with methamidophos, releasing the enzyme's active site. Our results suggest that IBTC, besides being an antioxidant and a promising antiatherogenic agent, is a non-toxic molecule for methamidophos poisoning treatment.

• 3-Thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-triazine improves neurite outgrowth and modulates MAPK phosphorylation and HSPs expression in H(2)O(2)-exposed PC12 cells.

  Authors: Fariba Khodagholi, Solaleh Khoramian Tusi, Shabnam Zeighamy Alamdary, Mohsen Amini, Niloufar Ansari

  Toxicology in vitro : an international journal published in association with BIBRA.

  Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. In this study we aimed to investigate the possible effect ofNeurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. In this study we aimed to investigate the possible effect of 3-thiomethyl-5,6-dimethoxyphenyl-1,2,4-triazine (TDMT) on H(2)O(2)-induced impairment of neurite outgrowth. We found that TDMT could improve neurite outgrowth and neurite complexity in H(2)O(2)-exposed PC12 cells. Moreover, we found elevated levels of Hsp-70 and suppressed level of Hsp-90 in TDMT-treated cells in the presence of H(2)O(2). As another important signaling pathways that play role in neuritogenesis, as well as apoptosis, we measured the level of phosphorylated and total MAPKs proteins, JNK, ERK and p38 MAPK. We found that TDMT inhibits oxidative stress-induced phosphorylation of MAPKs. Since HSPs and MAPKs are both involved in coping with environmental changes, it will not be surprising if they can modify or augment each other's activity. Neuroprotective effect of this compound could represent a promising approach for treatment of neurodegenerative diseases.

• The effect of acrylamide and nitric oxide donors on human mesenchymal progenitor cells.

  Authors: Lukasz Szewczyk, Justyna Ulańska, Marta Dubiel, Anna Maria Osyczka, Grzegorz Tylko

  Toxicology in vitro : an international journal published in association with BIBRA.

  We have examined the effects of nitric oxide donors and acrylamide on mesenchymal progenitor cell (hMPC) viability, programmed cell death (PCD) and differentiation. Acrylamide was examined at 0.5mMWe have examined the effects of nitric oxide donors and acrylamide on mesenchymal progenitor cell (hMPC) viability, programmed cell death (PCD) and differentiation. Acrylamide was examined at 0.5mM and 1.5mM concentrations, NOC-18 at 10μM and SNP at 100μM. Cell viability was assayed with MTS, PCD was determined by phosphatidylserine, caspase-9 and -3/7 and mitochondrial membrane potential assays, and osteogenic cell differentiation was evaluated by alkaline phosphatase activity (ALP) and mRNA levels for collagen type I, bone sialoprotein, ostepontin and osteocalcin. Serum-free hMPC cultures treated with 1.5mM acrylamide and SNP for 72h demonstrated reduced viability. PCD analyses revealed that SNP stimulated cells to necrosis in reactive species-dependent manner. Acrylamide led (1.5mM) to apoptosis independent of reactive species. Acrylamide and SNP reduced ALP activity and collagen type I mRNA levels but mRNA levels for bone sialoprotein and osteopontin increased in SNP treated cells and remained unchanged in acrylamide. Acrylamide had no effect on guanylate cyclase and cGMP osteogenic signaling pathway. The study suggests that acrylamide might impair bone development and remodeling upon acute or prolonged intoxication with this compound of mesenchymal cells.

• Co-cultures of enterocytes and hepatocytes for retinoid transport and metabolism.

  Authors: Carlotta Rossi, Barbara Guantario, Simonetta Ferruzza, Christiane Guguen-Guillouzo, Yula Sambuy, Maria Laura Scarino, Diana Bellovino

  Toxicology in vitro : an international journal published in association with BIBRA.

  Dietary retinoid bioavailability involves the interplay of the intestine (transport and metabolism) and the liver (secondary metabolism). To reproduce these processes in vitro, differentiated humanDietary retinoid bioavailability involves the interplay of the intestine (transport and metabolism) and the liver (secondary metabolism). To reproduce these processes in vitro, differentiated human intestinal Caco-2/TC7 cells were co-cultured with two hepatocyte cell lines. Murine 3A cells and the more highly differentiated human HepaRG hepatocytes were both shown to respond to β-carotene (BC) and retinol (ROH) treatment by secreting Retinol Binding Protein 4 (RBP4). In co-culture experiments, Caco-2/TC7 were differentiated on filter inserts and transferred for the time of the experiment to culture wells containing confluent 3A or differentiated HepaRG cells. Functionality of the co-cultures was assayed using as endpoints the retinol-dependent secretion of RBP4 and the retinoic acid-dependent induction of CYP26A1 in hepatocytes. BC and ROH added to intestinal Caco-2/TC7 induced a reduction in intracellular RBP4 levels in the underlying hepatocytes and its secretion into the medium. HepaRG hepatocytes were also shown to up-regulate the expression of CYP26A1 mRNA in response to retinoid treatment. This in vitro model represents a useful tool to analyze the absorption and metabolism of retinoids and could be further developed to investigate other dietary compounds and molecules of pharmacological interest.

• Hypochlorous acid-induced heme damage of hemoglobin and its inhibition by flavonoids.

  Authors: Lidia Gebicka, Ewa Banasiak

  Toxicology in vitro : an international journal published in association with BIBRA.

  Hypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive oxidizing and chlorinating agent of biologically relevant molecules. Hemoglobin (Hb), present in large amounts insideHypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive oxidizing and chlorinating agent of biologically relevant molecules. Hemoglobin (Hb), present in large amounts inside red blood cells, is the main target for HOCl in these cells. In this work heme damage of hemoglobin induced by hypochlorous acid in the absence and presence of some popular dietary flavonoids, catechin, epigallocatechin gallate and quercetin has been studied by stopped-flow spectrophotometry. Hypochlorous acid, being in a large molar excess to hemoglobin, initiates modifications of the heme group of oxy- as well as of methemoglobin, which eventually leads to heme damage. Flavonoids present at concentrations comparable with that of hemoglobin inhibit these processes. The kinetics of the reactions of the investigated flavonoids with HOCl has been also studied and the rate constants of the order of 10(5)M(-1)s(-1) have been found. It is concluded that under conditions used in this study the inhibition of Hb heme damage by flavonoids results from the competition of these compounds with hemoglobin towards HOCl and/or from the formation of Hb-flavonoid complex in which heme group is more resistant against HOCl-induced damage.

• The effect of a novel tobacco process on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter.

  Authors: R Combes, K Scott, D Dillon, C Meredith, K McAdam, C Proctor

  Toxicology in vitro : an international journal published in association with BIBRA.

  Some of the toxic effects of smoking have been attributed to the combustion of nitrogenous protein in tobacco. The effects of a treatment which reduces tobacco's protein nitrogen level, on the inSome of the toxic effects of smoking have been attributed to the combustion of nitrogenous protein in tobacco. The effects of a treatment which reduces tobacco's protein nitrogen level, on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter (PM), were measured. PMs were tested in the Neutral Red Uptake (NRU) test; the Salmonella mutagenicity assay (SAL); the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT). PMs from all of the cigarettes were cytotoxic and genotoxic. PM obtained from smoking treated tobacco, showed a small, consistent and statistically significant reduced mutagenicity (revertants/μg) in TA98 with post-mitochondrial supernatant (S9). No consistent quantitative or qualitative differences were detected in the other tests. The data are discussed in relation to published information on smoke chemistry obtained from cigarettes made of tobacco treated using this technique. The observations confirm that the method did not give rise to any new qualitative or quantitative cytotoxic or genotoxic effects, and may have reduced PM's bacterial mutagenicity in TA98 with S9. Further toxicity testing is warranted, to investigate the effects of the tobacco treatment in more detail and add to the data already obtained.

• Diesel exhaust particles impair platelet response to collagen and are associated with GPIbα shedding.

  Authors: Marc Forestier, Mohammad Al-Tamimi, Elizabeth Gardiner, Corinna Hermann, Sara C Meyer, Juerg H Beer

  Toxicology in vitro : an international journal published in association with BIBRA.

  OBJECTIVE: Air pollution with fine particulates (PM(10) and PM(2.5)) is associated with an increased incidence of cardiovascular events. The proposed mechanisms include indirect proinflammatory andOBJECTIVE: Air pollution with fine particulates (PM(10) and PM(2.5)) is associated with an increased incidence of cardiovascular events. The proposed mechanisms include indirect proinflammatory and procoagulant reactions involving activation of pulmonary macrophages, endothelial cells and the TNF/TF pathway, or direct procoagulant effects. Our laboratory has observed a reduction of the platelet responsiveness to collagen after exposure to diesel exhaust particles (DEP). HYPOTHESIS: DEP directly interfere with platelet-collagen interactions by selectively inducing the shedding of platelet signaling receptors via metalloproteinases, which would represent a novel mechanism for DEP action on platelets. METHODS: Citrated blood from healthy volunteers was exposed to highly standardized DEP at concentrations of 0.1, 2.5 and 5.0μg/ml or ultrafine carbon black (ufCB, 0.1μg/ml) and the plasmatic and platelet response was analysed. The closure times with the PFA-100 device and the platelet aggregation in response to a variety of agonists were monitored. Interleukins (IL)-1β and IL-8 levels were determined by ELISA and soluble P-selectin by the Luminex bead assay. Thrombin activity was measured as the endogenous thrombin potential (ETP) by fluorescence spectrometry. Soluble GPVI and GPIbα (glycocalicin) ectodomain fragments were measured by ELISA. ADAMTS13 activity was determined by a FRETS based assay and plasmin activity with Spectrozyme PL. RESULTS: Aggregation assays where platelets were treated with low dose DEP or ultrafine carbon black (ufCB) revealed a significantly increased response to low doses of collagen (p<0.05, n=5). At higher doses, however, collagen induced aggregation was suppressed by DEP treatment: at 2.5μg/ml, the inhibition was 34±12% (p<0.01, n=10). Aggregations with cross-linked collagen related peptide (CRPxl), convulxin and with the monoclonal antibody 9O12.2 (all known to specifically bind to and activate GPVI) were also diminished. Ristocetin, arachidonic acid and ADP responses were normal at all DEP concentrations. No cleavage of GPVI ectodomain was detected (soluble GPVI 27.8±3 vs. 28±4μg/ml mean±SEM, n=12); however increased plasma glycocalicin (GPIbα ectodomain) was detected upon diesel exposure (2.58±0.11 vs. 2.28±0.03μg/ml p<0.01, n=10). ADAMTS13 and plasmin activity remained unaffected by DEP under the conditions tested. Platelets were not activated by either DEP or ufCB as soluble P-selectin was insensitive to these. CONCLUSIONS: DEP specifically and directly interferes with platelet-collagen interactions. The functional consequences are biphasic and include enhance platelet aggregation at lower DEP concentrations and inhibition at a higher dose. Our data indicate that this interaction does not involve P-selectin or GPVI shedding. It is however associated with an increase in GPIb cleavage.

• Kinetic modeling of β-chloroprene metabolism: Probabilistic in vitro-in vivo extrapolation of metabolism in the lung, liver and kidneys of mice, rats and humans.

  Authors: Yuching Yang, Matthew W Himmelstein, Harvey J Clewell

  Toxicology in vitro : an international journal published in association with BIBRA.

  β-Chloroprene (chloroprene) is carcinogenic in inhalation bioassays with B6C3F1 mice and Fischer rats, but the potential effects in humans have not been adequately characterized. In order to provideβ-Chloroprene (chloroprene) is carcinogenic in inhalation bioassays with B6C3F1 mice and Fischer rats, but the potential effects in humans have not been adequately characterized. In order to provide a better basis for evaluating chloroprene exposures and potential effects in humans, we have explored species and tissue differences in chloroprene metabolism. This study implemented an in vitro-in vivo extrapolation (IVIVE) approach to parameterize a physiologically based pharmacokinetic (PBPK) model for chloroprene and evaluate the influence of species and gender differences in metabolism on target tissue dosimetry. Chloroprene metabolism was determined in vitro using liver, lung and kidney microsomes from male or female mice, rats, and humans. A two compartment PK model was used to estimate metabolism parameters for chloroprene in an in vitro closed vial system, which were then extrapolated to the whole body PBPK model. Two different strategies were used to estimate parameters for the oxidative metabolism of chloroprene: a deterministic point-estimation using the Nelder-Mead nonlinear optimization algorithm and probabilistic Bayesian analysis using the Markov Chain Monte Carlo technique. Target tissue dosimetry (average amount of chloroprene metabolized in lung per day) was simulated with the PBPK model using the in vitro-based metabolism parameters. The model-predicted target tissue dosimetry, as a surrogate for a risk estimate, was similar between the two approaches; however, the latter approach provided a measure of uncertainty in the metabolism parameters and the opportunity to evaluate the impact of that uncertainty on predicted risk estimates.

• Evaluation of molecularity of rate-limiting step of pore formation by antimicrobial peptides studied using mitochondria as a biosensor.

  Authors: Dinara Aliverdieva, Dmitry Mamaev, Leona Snezhkova, Christophor Sholtz

  Toxicology in vitro : an international journal published in association with BIBRA.

  Toxic agents, derived from bee or hornet venoms and from fungi - melittin, mastoparan, and alamethicin are able to permeabilize biological membranes. We studied the initial steps of pore formation byToxic agents, derived from bee or hornet venoms and from fungi - melittin, mastoparan, and alamethicin are able to permeabilize biological membranes. We studied the initial steps of pore formation by these peptides in rat liver mitochondria preparations (RLM) generating transmembrane potential (ΔΨ). RLM has been used as a potassium transmembrane current (PTC) sensor. The PTC induced in RLM depends linearly on the degree of steady-state activation of RLM respiration. The concentration order of such activation by melittin in a "potassium" incubation medium containing 6mM Mg(2+) was 2.01±0.15. In the case of mastoparan, the reaction order was 1.83±0.23. The first steady-state phase of activation of RLM respiration by alamethicin was not detected in "Tris" incubation medium; it appeared only after addition of KCl. The order of the reaction limiting such activation was 1.92±0.07. It is suggested that PTC in this phase is determined by the channels with the lowest degree of oligomerization formed by "dimmers". The ratio of equally active membrane concentrations of peptides obviously reflects the ratio of average lifetimes (ALT) for corresponding "dimers" (alamethicin and melittin, 38.5; mastoparan and melittin, 0.32). It is concluded that the results of this investigation may be useful for comparative testing of perspective pharmaceuticals.

• 4-(2-Hydroxypropan-2-yl)-1-methylcyclohexane-1,2-diol prevents xenobiotic induced cytotoxicity.

  Authors: Anup Srivastava, L Jagan Mohan Rao, T Shivanandappa

  Toxicology in vitro : an international journal published in association with BIBRA.

  Currently there is a great deal of interest in the study of natural compounds with free radical scavenging activity because of their potential role in maintaining human health and preventingCurrently there is a great deal of interest in the study of natural compounds with free radical scavenging activity because of their potential role in maintaining human health and preventing diseases. In this paper, we report the antioxidant and cytoprotective properties of 4-(2-hydroxypropan-2-yl)-1-methylcyclohexane-1,2-diol (HPMCD) isolated from the aqueous extract of Decalepis hamiltonii roots. Our results show that HPMCD is a potent scavenger of superoxide (O(2)(-)), hydroxyl (()OH), nitric oxide (()NO), and lipid peroxide (LOO()) physiologically relevant free radicals with IC(50) values in nmolar (56-582) range. HPMCD also exhibited concentration dependent secondary antioxidant activities like reducing power, metal chelating activity, and inhibition of protein carbonylation. Further, HPMCD at nmolar concentration prevented CuSO(4)-induced human LDL oxidation. Apart from the in vitro free radical scavenging activity HPMCD demonstrated cytoprotective activity in primary hepatocytes and ehrlich ascites tumor (EAT) cells against oxidative stress inducing xenobiotics. The mechanism of cytoprotective action involved maintaining the intracellular glutathione (GSH), scavenging of reactive oxygen species (ROS), and inhibition of lipid peroxidation (LPO). Based on the results it is suggested that HPMCD is a novel bioactive molecule with health implications in both prevention and amelioration of diseases involving oxidative stress as well as in the general well being.

• Evaluating biotoxicity with fibroblasts derived from human embryonic stem cells.

  Authors: Xiaoying Wang, Shenglin Li, Tong Cao, Xin Fu, Guangyan Yu

  Toxicology in vitro : an international journal published in association with BIBRA.

  To investigate the use of differentiated fibroblasts from human embryonic stem cells as a cellular model for cytotoxicity and genotoxicity screening. The EBf-H9 cells were derived from humanTo investigate the use of differentiated fibroblasts from human embryonic stem cells as a cellular model for cytotoxicity and genotoxicity screening. The EBf-H9 cells were derived from human embryonic stem cells (H9) via embryonic body (EB) and treated with Sodium fluoride (NaF) and Formaldehyde (FA). Proliferation, specific gene and protein expression and karyotype of cells were analyzed by MTT assay, RT-PCR, immunocytochemistry and karyotype analysis, respectively. Cytotoxicity was detected by MTT assay and flow cytometry, and genotoxicity was studied by micronucleus test (MNT), sister chromatid exchange (SCE) and comet assay. EBf-H9s were spindle-shaped with a diploid karyotype. They expressed the fibroblast markers prolyl 4-hydroxylase β and vimentin but did not express Oct-4 and Sox-2, and decreased expression of Nanog. The proliferation of EBf-H9 and murine L929 cells was inhibited by sodium fluoride (NaF) and formaldehyde (FA), and the cell cycle was arrested in different phases with the treatments. In genotoxicity assays with NaF and FA, positive responses were detected in human EBf-H9s comparable to those in the murine L929 cell line. EBf-H9 may be a suitable new cell source for toxicity research on biomaterials and other agents.

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