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Tobacco smoke is a complex mixture with over 8700 identified constituents. Smoking causes many diseases including lung cancer, cardiovascular disease, and chronic obstructive pulmonary disease. However, the mechanisms of how cigarette smoke impacts disease initiation or progression are not well understood and individual smoke constituents causing these effects are not generally agreed upon. The studies reported here were part of a series of investigations into the contributions of selected smoke constituents to the biological activity of cigarette smoke. In vitro cytotoxicity measured by the neutral red uptake (NRU) assay and in vitro mutagenicity determined in the Ames bacterial mutagenicity assay (BMA) were selected because these assays are known to produce reproducible, quantitative results for cigarette smoke under standardized exposure conditions. In order to determine the contribution of individual cigarette smoke constituents, a fingerprinting method was developed to semi-quantify the mainstream smoke yields. For cytotoxicity, 90% of gas vapor phase (GVP) cytotoxicity of the Kentucky Reference cigarette 1R4F was explained by 3 aldehydes and 40% of the 1R4F particulate phase cytotoxicity by 10 smoke constituents, e.g., hydroquinone. In the microsuspension version of the BMA, 4 aldehydes accounted for approximately 70% of the GVP mutagenicity. Finally, the benefits of performing such studies along with the difficulties in interpretation in the context of smoking are discussed.

Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses. Goals of this study: 1) demonstrate functionality of the VITROCELL® VC10® system by installation, operational and performance qualification, 2) develop and validate a cellular system for assessing cytotoxicity following whole smoke exposure and 3) assess the whole smoke NRU assay sensitivity for statistical differentiation between a reference combustible cigarette (3R4F) and a primarily “heat-not-burn” cigarette (Eclipse). Results The VITROCELL® VC10® provided consistent generation and delivery of whole smoke; exposure-related changes in in vitro cytotoxicity were observed with reproducible IC50 values; comparative analysis showed that the heat-not-burn cigarette was significantly (P

Phomoxanthone A (PhoA) is a compound isolated from the endophytic fungus Phomopsis longicolla, associated with marine algae Bostrychia radicans. Although this metabolite was previously described regarding its high biological potential, there are no reports concerning the effects of this compound on DNA integrity. This study aimed to evaluate, in lymphocytes and promyelocytic leukemia HL60 cells, the cytotoxicity of this compound through MTT and neutral red (NR) assays, as well as its genotoxicity and mutagenicity by alkaline comet assay and cytokinesis-block micronucleus cytome assay (CBMN-Cyt), respectively. Cells were treated with PhoA concentrations ranging from 0.01 to 100.0μg/mL, and the results show that this molecule did not exhibit cytotoxicity, genotoxicity or mutagenicity in lymphocytes at any tested concentration. Furthermore, PhoA was highly cytotoxic, genotoxic and mutagenic to HL60 cells, establishing a differential response of this natural product in normal and cancer cells. PhoA was highly selective towards HL60 compared to lymphocytes, causing no damage in the latter cell line, suggesting that this compound could be a promising compound in antitumoral drug development.

The use of electronic vaping products (EVPs) continues to increase worldwide among adult smokers in parallel with accumulating information on their potential toxicity and relative safety compared to tobacco smoke. At this time, in vitro assessments of many widely available EVPs are limited. In this study, an in vitro battery of established assays was used to examine the cytotoxic (Neutral red uptake), genotoxic (In vitro micronucleus) and mutagenic (Bacterial reverse mutation) responses of two commercial EVPs (blu GO™ disposable and blu PLUS+™ rechargeable) when compared to smoke from a reference cigarette (3R4F). In total, 12 commercial products were tested as e-liquids and as aerosols. In addition, two experimental base liquids containing 1.2% and 2.4% nicotine were also assessed to determine the effect of flavour and nicotine on all three assays. In the bacterial reverse mutation (Ames) and in vitro micronucleus (IVM) assays, exposures to e-liquids and EVP aerosols, with and without nicotine and in a range of flavourings, showed no mutagenic or genotoxic effects compared to tobacco smoke. The neutral red uptake (NRU) assay showed significantly reduced cytotoxicity (P .05) for whole undiluted EVP aerosols compared to tobacco smoke, which by contrast was markedly cytotoxic even when diluted. The reduced in vitro toxicological responses of the EVPs add to the increasing body of scientific weight-of-evidence supporting the role of high-quality EVPs as a harm reduction tool for adult smokers.

Submitted by Luana Priscila Costa (luana@reitoria.unesp.br) on 2020-11-25T18:58:43Z No. of bitstreams: 1 silva_ndg_preprint_soro_interference.pdf: 851822 bytes, checksum: e94ff4ddbf0c7fb3b9018ab7069257dd (MD5) Approved for entry into archive by Luana Priscila Costa (luana@reitoria.unesp.br) on 2020-11-25T18:59:46Z (GMT) No. of bitstreams: 1 silva_ndg_preprint_soro_interference.pdf: 851822 bytes, checksum: e94ff4ddbf0c7fb3b9018ab7069257dd (MD5) Made available in DSpace on 2020-11-25T18:59:46Z (GMT). No. of bitstreams: 1 silva_ndg_preprint_soro_interference.pdf: 851822 bytes, checksum: e94ff4ddbf0c7fb3b9018ab7069257dd (MD5) Previous issue date: 2020-06 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Goethite (α-FeOOH) brings important perspectives in environmental remediation, as, due to its physicochemical properties, this iron oxide can adsorb a wide variety of compounds, including glyphosate. This study aimed to evaluate the effects of goethite nanoparticles (NPs), glyphosate (Gly), Roundup® (Rd), and co-exposures (Gly + NPs and Rd + NPs) on zebrafish liver cell line (ZFL). ZFL cells were exposed to NPs (1, 10, and 100 mg L−1), Gly (3.6 mg L−1), Rd (10 mg L−1), and co-exposures (Gly + NPs and Rd + NPs), or only to saline for 1, 6, and 12 h. Cell viability was assessed by Trypan blue, MTT, and neutral red assays. The generation of reactive oxygen species and total antioxidant capacity were also determined, while genotoxicity was quantified by the comet assay. Both NPs and Rd in isolation produced cytotoxic effects at 6 h and genotoxic effects at 1 and 6 h. Rd + NPs resulted in synergistic effects, intensifying the toxicity. Cells exposed to Gly did not present toxic effects and Gly + NPs resulted in the suppression of toxic effects observed for NPs. The presence of other components in Roundup® seems to favor its toxicity compared to the active ingredient. In conclusion, according to the in vitro model, the concentrations used were not safe for the ZFL lineage. Preprint Universidade Estadual de Londrina – UEL, Londrina, Paraná, Brazil Universidade Estadual Paulista – UNESP, Sorocaba, São Paulo, Brazil FAPESP: 2017/21004-5

To assess photoxicity, several in vitro methods using different cellular models have been developed for preclinical testing. Over prediction of the in vivo photosafety hazard has been however appointed. Herein, we describe the implementation and validation of an in vitro methodology for phototoxicity evaluation based on the 3T3 neutral red uptake phototoxicity test using the HaCaT human keratinocyte cell line, and UVA/UVB radiation. Known positive (5-methoxypsoralen, chlorpromazine, and quinine) and negative (acetyl salicylic acid, hexachlorophene, and sodium lauryl sulphate) controls were tested together with a set of chemical currently used in cosmetic/pharmaceutical formulations. Apart from the advantage of using a cell line of human origin, these cells were generally more resistant to the cytotoxic effects of the test substances relative to the 3T3 mouse fibroblasts when exposed to an UVA irradiation dose of 1.7 mW/cm2. Therefore, this HaCaT NRU assay provides a more realistic experimental model that overcomes the over/high sensitivity frequently noted with the 3T3 NRU assay and that is more consistent with the human in vivo situation. Using a more representative method can prevent time-consuming and expensive in vivo testing in both animal models and humans that can significantly delay the clinical development of new chemicals.

Toxicological interaction represents a challenge to toxicology, particularly for novel contaminants. There are no data whether silver nanoparticles (AgNPs), present in a wide variety of products, can interact and modulate the toxicity of ubiquitous contaminants, such as nonessential metals. In the current study, we investigated the toxicological interactions of AgNP (size = 1–2 nm; zeta potential = − 23 mV), cadmium and mercury in human hepatoma HepG2 cells. The results indicated that the co-exposures led to toxicological interactions, with AgNP + Cd being more toxic than AgNP + Hg. Early (2–4 h) increases of ROS (DCF assay) and mitochondrial O 2[rad] − levels (Mitosox® assay) were observed in the cells co-exposed to AgNP + Cd/Hg, in comparison to control and individual contaminants, but the effect was partially reverted in AgNP + Hg at the end of 24 h-exposure. In addition, decreases of mitochondrial metabolism (MTT), cell viability (neutral red uptake assay), cell proliferation (crystal violet assay) and ABC-transporters activity (rhodamine accumulation assay) were also more pronounced in the co-exposure groups. Foremost, co-exposure to AgNP and metals potentiated cell death (mainly by necrosis) and Hg 2 + (but not Cd 2 +) intracellular levels (ICP-MS). Therefore, toxicological interactions seem to increase the toxicity of AgNP, cadmium and mercury.

Cell-based assays are key tools in drug safety assessment. However, they usually provide only limited information about time-kinetics of a toxic effect and implementing multiple measurements is often complex. To overcome these issues we established an impedance-based approach which is able to differentiate cytostatic from cytotoxic drugs by recording time-kinetics of compound-effects on cells. NIH 3T3 fibroblasts were seeded on xCELLigence® E-plates and impedance was continuously measured over 5 days. The obtained results reflected cytotoxicity and cell proliferation, as confirmed by neutral red uptake in vitro. Based on known toxicants, we established an algorithm able to discriminate cytostatic, cytotoxic and non-toxic compounds based on the shape of the impedance curves. Analyzing impedance curve patterns of additional 37 compounds allowed the identification and differentiation of these distinct effects as results correlated well with previous in vivo findings. We show that impedance-based real-time cell analysis is a convenient tool to characterize and discriminate effects of compounds on cells in a time-dependent and label-free manner. The presented impedance assay could be used to further characterize toxicities observed in vivo or in vitro. Due to the ease of performance it may also be a suitable screening tool.

Tobacco smoke contains more than 5600 constituents, of which approximately 150 are toxicants. This paper describes the activities in the Neutral Red uptake (NRU) assay, the Salmonella mutagenicity test (SAL), the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT) of Particulate Matter (PM) obtained from experimental cigarettes (ECs), designed to produce reduced levels of toxicants. The designs included tobacco substitute sheet (TSS) containing glycerol, which dilutes toxicants in smoke, or the incorporation of blend-treated (BT) tobacco to reduce the levels of nitrogenous toxicant precursors and some polyphenols. All samples were cytotoxic in the NRU, however TSS reduced PM cytotoxicity in this assay. All PMs were mutagenic in the SAL, MLA and IVMNT. Reductions in bacterial mutagenicity were observed in the SAL, for cigarettes with BT tobacco, compared with their respective controls. The quantitative changes in bacterial mutagenicity could be explained by analytical chemistry data on smoke generated from the ECs used in the study. These observations, and the absence of consistent qualitative differences in the activities of the experimental, control and reference cigarettes, suggest that reduced toxicity cigarettes, as measured by the tests described in this paper, may be developed without introducing any additional cytotoxic or genotoxic hazards, but the impact of this on human health risks remains unknown.

When in vitro test systems are evaluated for assessment of the toxicity of chemical compounds, particular efforts are made to mimic the in vivo reality as close as possible. Cellular models with appropriate metabolic competence, i.e. with the potency to biotransform chemical compounds, are considered crucial since some metabolites have a different toxicity than their parent compounds. In this study a cell based in vitro test system is proposed to investigate the basal cytotoxicity of several reference chemicals. Both metabolic competent HepaRG cells and cells with no or low hepatic enzyme activity (undifferentiated HepaRG and proliferating HepG2) were used. The classic Neutral Red Uptake (NRU) assay proved to be robust and reliable to be applied as viability assay. The test was performed on a robotic platform, which enabled fully automated and simultaneous screening of the compounds. The outcome of these tests grouped the tested compounds in three categories following their detoxification effect (benzo(a)pyrene, valproic acid), their bio-activation effect (aflatoxin B1) and their specific effect on inhibition of cell proliferation (cycloheximide, sodium lauryl sulphate, atropine sulphate monohydrate, acetylsalicylic acid).

Intracellular Ca(2+) increase is a common feature of multiple cellular pathways associated with receptor and channel activation, mediator secretion and gene regulation. We investigated the possibility of using this Ca(2+) signal as a biomarker for a reaction to chemical irritants of normal human keratinocytes (NHK) in submerged primary cell culture. We tested 14 referenced chemical compounds classified as strong (seven), weak (four) or non- (three) irritants in acute irritant contact dermatitis. We found that the strong irritant compounds tested at 20-40 mM induced an intracellular Ca(2+) increase measurable by spectrofluorimetry in an automated test. Weak and non-irritant compounds however did not increase intracellular Ca(2+) concentration. We further investigated the mechanisms by which the amine heptylamine, classified as a R34 corrosive compound, increases intracellular Ca(2+). Heptylamine (20mM) induced an ATP release that persisted in the absence of intra- and extra-cellular Ca(2+). In addition, we found that this ATP activates NHK purinergic receptors that subsequently cause the increase in intracellular Ca(2+) from sarcoplasmic reticular stores. We conclude that measuring the intracellular Ca(2+) concentration in NHK is a suitable and easy way of determining any potential reaction to soluble chemical compounds.

Silver nanoparticles (AgNPs) are among the most widely commercialised engineered nanomaterials, because of their antimicrobial properties. They are already commonly used in medical devices, household products and industry. Concerns have been raised about potential adverse health effects due to increasing dispersion of AgNPs in the environment. The present study examined the cytotoxic effects of spherical, citrate-coated AgNPs (10, 50 and 100 nm) in rat brain endothelial (RBE4) cells and investigated whether the observed effects can be explained by the intrinsic toxicity of the particles or the silver ions released from the particles. The results indicated that exposure of RBE4 cells to AgNPs lead to significant reduction in dye uptake as measured with the Neutral red (NR) assay. The effect was found to be related to particle size, surface area, dose and exposure time. In contrast, silver ions increased NR uptake (ca. 10%) in RBE4 cells after 1h, while a reduction in NR uptake was observed after 24h exposure at high concentrations (20-30 μM). Colony formation, as an indicator of proliferation ability, was completely inhibited by AgNPs at concentrations higher than 1 μg/ml. Silver ions had less effect on the colony formation of RBE4 cells than AgNPs.

An in vitro model simulating the intestinal barrier for efficacy and drug interaction testing of mycotoxin detoxifying agents was developed using Transwell® cell culture inserts. Intestinal porcine epithelial cells derived from the jejunum of piglets were exposed to DON and a mycotoxin binder (efficacy testing) or exposed to tylosin and a mycotoxin binder (drug interaction testing). Active carbon and bentonite were used in the efficacy and drug interaction trials, respectively, to validate the developed model. The evaluated parameters were passage of DON and tylosin through the epithelial monolayer, the integrity of the monolayer by measurements of the trans-epithelial electrical resistance and the viability of the monolayer using the neutral red assay. In the efficacy model it was shown that active carbon effectively bound DON at both non-cytotoxic and cytotoxic concentrations of DON, respectively 0.5 and 1 μg/mL. Moreover, the negative effects of DON at cytotoxic concentrations on cellular viability and integrity were completely offset. A commercially available modified gluco-mannan binder was also tested and it was able to partly reduce the negative effects on these latter parameter. Moreover, it reduced the transepithelial passage of DON with 37% to 57% compared to active carbon, at both cytotoxic and non-cytotoxic concentrations of DON. In our drug interaction model, the interaction between tylosin and mycotoxin binders was investigated as some authors suggest binding of macrolide antibiotics to bentonite clays. Indeed, a bentonite clay showed decreased passage of tylosin through the epithelial monolayer, indicating binding of tylosin by bentonite. This indicates that the combined use of bentonite and tylosin in the feed could lead to therapy failure. The modified gluco-mannan binder did not alter the passage of tylosin significantly, indicating safe combined use.

The aim of this study was to evaluate the in vitro skin phototoxicity of cosmetic formulations containing photounstable and photostable UV-filters and vitamin A palmitate, assessed by two in vitro techniques: 3T3 Neutral Red Uptake Phototoxicity Test and Human 3-D Skin Model In Vitro Phototoxicity Test. For this, four different formulations containing vitamin A palmitate and different UV-filters combinations, two of them considered photostable and two of them considered photounstable, were prepared. Solutions of each UV-filter and vitamin under study and solutions of four different combinations under study were also prepared. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (3T3-NRU-PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D-PT). Avobenzone presented a pronounced phototoxicity and vitamin A presented a tendency to a weak phototoxic potential. A synergistic effect of vitamin A palmitate on the phototoxicity of combinations containing avobenzone was observed. H3D-PT results did not confirm the positive 3T3-NRU-PT results. However, despite the four formulations studied did not present any acute phototoxicity potential, the combination 2 containing octyl methoxycinnamate (OMC), avobenzone (AVB) and 4-methylbenzilidene camphor (MBC) presented an indication of phototoxicity that should be better investigated in terms of the frequency of photoallergic or chronic phototoxicity in humans, once these tests are scientifically validated only to detect phototoxic potential with the aim of preventing phototoxic reactions in the general population, and positive results cannot predict the exact incidence of phototoxic reactions in humans.

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 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.

Titanium dioxide nanoparticles (TiO(2)-NPs) induced cytotoxicity and DNA damage have been investigated using human amnion epithelial (WISH) cells, as an in vitro model for nanotoxicity assessment. Crystalline, polyhedral rutile TiO(2)-NPs were synthesized and characterized using X-ray diffraction (XRD), UV-Visible spectroscopy, Fourier transform infra red (FTIR) spectroscopy, and transmission electron microscopic (TEM) analyses. The neutral red uptake (NRU) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assays revealed the concentration dependent cytotoxic effects of TiO(2)-NPs (30.6nm) in concentration range of 0.625-10μg/ml. Cells exposed to TiO(2)-NPs (10μg/ml) exhibited significant reduction (46.3% and 34.6%; p0.05) in catalase activity and glutathione (GSH) level, respectively. Treated cells showed 1.87-fold increase in intracellular reactive oxygen species (ROS) generation and 7.3% (p0.01) increase in G(2)/M cell cycle arrest, as compared to the untreated control. TiO(2)-NPs treated cells also demonstrated the formation of DNA double strand breaks with 14.6-fold (p0.05) increase in Olive tail moment (OTM) value at 20μg/ml concentration, vis-à-vis untreated control, under neutral comet assay conditions. Thus, the reduction in cell viability, morphological alterations, compromised antioxidant system, intracellular ROS production, and significant DNA damage in TiO(2)-NPs exposed cells signify the potential of these NPs to induce cyto- and genotoxicity in cultured WISH cells.

The in vitro effects of flurochloridone (FLC) and its formulations Twin Gold Pack® (25% a.i.) and Rainbow® (25% a.i.) were evaluated on Chinese hamster ovary (CHO-K1) cells by genotoxicity [sister chromatid exchange (SCE)] and cytotoxicity [cell-cycle progression, proliferative rate index (PRI), mitotic index (MI), MTT, and neutral red] end points. Cells were treated for 24. h within the 0.25-15 μg/ml concentration range. FLC and Twin Pack Gold® induced a significant and equivalent increase in SCEs regardless of the concentration. Rainbow®-induced SCEs at concentrations higher than 2.5 μg/ml; however, the increases were always lower than those induced by FLC and Twin Pack Gold®. For all compounds, the PRI decreased as a function of the concentration titrated into cultures. Whereas only the highest FLC and Twin Pack Gold® concentrations induced a significant reduction of the MI, all tested Rainbow® concentrations induced MI inhibition. Overall, the results demonstrated that although all compounds were not able to reduce the lysosomal activity, the mitochondrial activity was diminished when the highest concentrations were employed. These observations represent the first study analyzing the genotoxic and cytotoxic effects exerted by FLC and two formulated products on mammalian cells in vitro, at least on CHO-K1 cells. Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Malignant mesothelioma (MMe) is a lethal tumor arising from the mesothelium of serous cavities as a result of exposure to asbestos. Current clinical standards consist of combined treatments, but an effective therapy has not been established yet and there is an urgent need for new curative approaches. Ascorbate is a nutrient that is also known as a remedy in the treatment of cancer. In the present study, we have tested the cytotoxicity of ascorbate to MMe cells in combination with drugs used in MMe therapy, such as cisplatin, etoposide, gemcitabine, imatinib, paclitaxel, and raltitrexed, as well as with promising antitumor compounds like taurolidine, α-tocopherol succinate, and epigallocatechin-3-gallate (EGCG). Dose-response curves obtained for each compound by applying the neutral red uptake (NRU) assay to MMe cells growing in vitro, allowed to obtain IC50 values for each compound used singularly. Thereafter, NRU data obtained from each ascorbate/drug combination were analyzed through Tallarida's isobolograms at the IC50 level (Tallarida, 2000), revealing synergistic interactions for ascorbate/gemcitabine and ascorbate/EGCG. These results were further confirmed through comparisons between theoretical additivity IC50 and observed IC50 from fixed-ratio dose-response curves, and over a broad range of IC levels, by using Chou and Talalay's combination index (Chou and Talalay, 1984). Synergistic interactions were also shown by examining apoptosis and necrosis rates, using the caspase 3 and lactic dehydrogenase assays, respectively. Hence, data indicate that ascorbate/gemcitabine and ascorbate/EGCG affect synergistically the viability of MMe cells and suggest their possible use in the clinical treatment of this problematic cancer.

The human skin equivalent model (HSEM) is well known as an attractive alternative model for evaluation of dermal toxicity. However, only limited data are available on the usefulness of a HSEM for nanotoxicity testing. This study was designed to investigate cutaneous toxicity of polystyrene and TiO2 nanoparticles using cultured keratinocytes, a HSEM, and an animal model. In addition, we also evaluated the skin sensitization potential of nanoparticles using a local lymph node assay with incorporation of BrdU. Findings from the present study indicate that polystyrene and TiO2 nanoparticles do not induce phototoxicity, acute cutaneous irritation, or skin sensitization. Results from evaluation of the HSEMs correspond well with those from animal models. Our findings suggest that the HSEM might be a useful alternative model for evaluation of dermal nanotoxicity.

In vitro cytotoxicity tests are typically carried out with transformed, immortalized cell lines or primary cells. Immortalized cells are readily available and easily maintained, although they usually show anomalous behavior and phenotypes, which do not reflect the mechanisms observed in their normal homologous cells. Primary cells are indeed considered a better option as model systems for predicting toxicological behavior, although they are limited in quantity and suffer from batch-to-batch variation due to the need to isolate them freshly for each study. In particular, human Mesenchymal Stem Cells (hMSCs) have never been adopted in order to develop in vitro model systems for acute toxicity tests of chemicals. Therefore, the aim of this study was to verify the possibility of using hMSCs as an alternative method to estimate in vivo starting dose for acute toxicity. As suggested by ICCVAM, 12 reference chemicals were assessed in the present study and a Neutral Red Uptake assay was performed. It is shown for the first time that MSCs isolated from human bone marrow can be confidently used in this area of toxicology. MSCs represent a good promise for the development of in vitro human assays and could ultimately replace, improve or overtake current predictive models in toxicology.

Recently, the environmental residues of polybrominated diphenyl ethers (PBDEs) have markedly increased. In particular, the levels of certain PBDE congeners in fish have raised concern regarding potential risks associated with dietary PBDEs exposures. However, little is known regarding PBDE-mediated cell injury in relevant in vitro fish cell models. In this study, the cytotoxic effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209) on RTG-2 cells were investigated. RTG-2 cells were incubated with different concentrations of BDE-47 and BDE-209 (1-100 mu M) for 72 h, and a set of bioassays were conducted to measure: cell viability (evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and neutral red (NR) uptake), lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) formation and cell apoptosis. The results showed that BDE-47 and BDE-209 inhibited the cells viability, increased LDH leakage, and induced cell apoptosis in time and concentration-dependent manner. All significant effects were observed at concentrations of 12.5 mu M and above for BDE-47 and 25 mu M and above for BDE-209 (P < 0.05). At the concentration of 100 mu M BDE-47 and BDE-209, the cell viability of the exposed cells dropped to about 40% and 50% of the control, and the apoptotic rates were 52.6% and 34.6%, respectively. After 12 h exposure, a concentration-dependent increases of BDE-47 and BDE-209 (12.5-100 mu M) in ROS formation were observed. Collectively, the results of cell viability. LDH leakage, cell apoptosis and ROS formation demonstrated that the toxic mechanism of PBDEs on RTG-2 might be mediated by oxidative stress. (C) 2010 Elsevier Ltd. All rights reserved.

Cigarette smoking is associated with chronic obstructive pulmonary disease (COPD)--a term encompassing chronic lung inflammation, chronic bronchitis and emphysema. Goblet cell hyperplasia is a characteristic feature of the lung epithelium in COPD patients contributing to the overproduction of airway mucus, including mucin MUC5AC. Using an in vitro model of differentiated lung epithelium we have investigated morphological and cellular changes in response to interleukin (IL)-13 (2.5-20 ng/ml), cigarette smoke total particulate matter (TPM; 0.31-20 microg/ml) and three mainstream cigarette smoke constituents: acrolein, formaldehyde and acetaldehyde (all at 0.001-1 microM) over 28 days during differentiation (agents replaced daily Monday-Friday). Control cultures of human bronchial epithelial cells (HBECs) underwent mucociliary differentiation producing a columnar epithelium containing goblet, ciliated and basal cells. Non-cytotoxic doses of IL-13 induced a significant increase in the percentage of MUC5AC positive cells. Using both flow cytometry and immunocytochemical techniques for identification of MUC5AC positive cells, TPM treatment induced an increase in MUC5AC positive cells, becoming maximal at 5 microg/ml. Acrolein treatment also increased the percentage of MUC5AC positive cells. However, formaldehyde or acetaldehyde had little effect. This study demonstrates that lung toxicants can induce a profound effect on cellular differentiation in an in vitro model of the human lung epithelium.

Vanadium oxide nanostructures have potential uses for electrochemistry and catalysis, yet little is known about their toxicology. In this study, cultured human colon carcinoma cells (Caco-2) were exposed to vanadium oxide and their viability assessed with the neutral red assay. Cells exposed to either vanadium oxide (powdered form) or ethylene diamine intercalated vanadium oxide (enH2)V7O16 demonstrated no significant reduction in viability after twenty-four hours, yet cells exposed to vanadium oxide nanotubes demonstrated a significant loss in viability after four hours. The physical size and structure of the nanotubes may play an important role in their cytotoxic effects, and the safety of using such nanomaterials must be considered.

Dendritic polymer nanoparticles such as polyamidoamine dendrimers (PAMAM) show exciting potential for biomedical applications. While the potential commercial applications of such dendrimers have received considerable attention, little is known about their possible adverse effects on both humans and the environment. In this study, the in vitro cytotoxicocity of full generation PAMAM dendrimers to two mammalian cell lines was investigated. Generations G4, G5 and G6 were chosen. Metabolic, lysosomal and mitochondrial activities were evaluated after 24 h exposure. Long term toxicity was evaluated using the clonogenic assay. Particle size and zeta potential were measured in all media. In culture medium, the particle size was largely unchanged from that observed in phosphate buffer. The zeta potential changed significantly, however, from positive in deionized water to negative in culture medium. A linear correlation was found between the change in zeta potential of the dendrimers in media and their surface area measured in phosphate buffer. The interaction of the dendrimer nanoparticles with 5% FBS supplemented media was also studied using UV/visible spectroscopy. The data suggest significant interaction of nanoparticles with FBS and other media components which increased with dendrimer generation. The toxicity also correlated linearly with the zeta potential and notably the change in zeta potential in the media, further pointing towards indirect toxic mechanisms. A trend of generation dependent toxic response and interaction of the dendrimers with the cell culture media was observed which may lay the basis of structure activity relationships.

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC(50) values obtained on the MTT and NR assays ranged from 0.69 to 79.40 microM and 4.40 to 108.76 microM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H(2)-DCFDA was measured. The relative intensity of fluorescence from cells incubated with BEA, PAT and ZEA was approximately 4-, 7- and 4-fold higher in comparison with control cells at 0 min, respectively. Lipid peroxidation induced by ROS generation was assessed using the thiobarbituric acid reactive substances (TBARS) method for 2, 24 and 48 h. The malondialdehyde (MDA) production was increased with BEA and PAT exposure in a concentration- and time-dependent manner. MDA was not increased after 1 and 5 microM ZEA exposures for 2h, but was slightly increased at 50 microM. In conclusion, PAT was the most cytotoxic mycotoxin to CHO-K1 cells, followed by BEA and ZEA. Mycotoxins reduce cell viability correlated with the increases of ROS generation and MDA formation in concentration- and time-dependent manner. These data suggested that cytotoxicity and ROS generation are mechanisms of mycotoxins mediated toxicity.

The use of novel materials on the modern battlefield, both in military munitions as well as in Improvised Explosive Devices, opens the possibility of wounds with embedded fragments whose health effects and toxicity characteristics have not been fully investigated, if at all. The costly and time-consuming nature of standard two-year lifespan studies prohibits the testing of many materials. In this report, we describe an in vitro system for rapidly assessing potential toxicity of metals and metal mixtures. Using rat L6 and mouse C2C12 skeletal muscle cells and tests for cellular viability, we have shown that two militarily relevant tungsten alloy mixtures (W/Ni/Co and W/Ni/Fe) significantly decreased the metabolic viability of rat L6 cells, whereas the viability of mouse C2C12 cells was not affected by W/Ni/Co and only slightly affected by W/Ni/Fe. In addition, viability assessed through lysosomal uptake of neutral red dye was not affected by either mixture in either cell line indicating that the mitochondria may be the target organelle of these unique metal mixtures. Development of this in vitro screening system may provide a procedure by which the potential toxicities of embedded metal fragments can be rapidly assessed.

Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extra-cellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.

Treatment of human oral squamous carcinoma HSC-2 cells and normal GN46 fibroblasts with theaflavin-3,3'-digallate (TF-3), a polyphenol in black tea, showed a concentration and time dependent inhibition of growth, with the tumor cells more sensitive than the fibroblasts. In buffer and in cell culture medium, TF-3 generated reactive oxygen species, with lower levels detected in buffer amended with catalase and superoxide dismutase, indicating the generation of hydrogen peroxide and superoxide, respectively, and suggesting that TF-3 may be an inducer of oxidative stress. The toxicity of TF-3 was decreased in the presence of catalase, pyruvate, and divalent cobalt, all scavengers of reactive oxygen species, but was potentiated in the presence of diethyldithiocarbamate, an inhibitor of superoxide dismutase. The intracellular level of glutathione in HSC-2 cells was lessened after a 4-h exposure to 250 and 500 microM TF-3. However, for GN46 fibroblasts, a 4-h exposure to 250 microM TF-3 stimulated, but to 500 microM TF-3 lessened, intracellular glutathione. Treatment of the cells with the glutathione depleters, 1,3-bis(2-chloroethyl)-N-nitrosourea, 1-chloro-2,4-dinitrobenzene, and d,l-buthionine-[S,R]-sulfoximine potentiated the toxicity of TF-3. Induction of apoptotic cell death in HSC-2 cells treated with TF-3 was noted by apoptotic cell morphologies, by TUNEL staining, by PARP cleavage, and by elevated activity of caspase-3. Apoptosis was not noted in GN46 fibroblasts treated with TF-3.

The effects of the addition of ammonium magnesium phosphate (AMP) to the paper of an electrically heated cigarette (EHC) prototype on smoke composition and toxicity were quantified and the underlying mechanisms investigated. Smoke from EHC prototypes with and without AMP and from conventional cigarettes, i.e. the University of Kentucky Standard Reference Cigarette 1R4F and eight American-blend market cigarettes, was compared. Endpoints for comparison were smoke chemistry, where toxic constituents were measured, cytotoxic activity, as measured in murine fibroblasts embryo cells by the Neutral Red Uptake Assay, and genotoxic activity, as measured in bacteria by the Salmonella Reverse Mutation Assay and in murine lymphoma cells by the TK Assay. The addition of AMP to the EHC led to a reduction of toxic substances and toxicological activity of approximately 30% compared to the EHC without AMP. Compared to the conventional cigarettes, the EHC with AMP showed reductions of 75-90%. Smoke from the EHCs generated in nitrogen atmospheres supplemented with different concentrations of ammonia and oxygen was assayed for its in vitro cytotoxicity and genotoxicity. The results indicate that the ammonia released by AMP at the heating site of the EHC is responsible for the reductions in cytotoxicity and mutagenicity for the EHC with AMP compared with the EHC without AMP. Thus, while the EHC approach distinctly reduces toxic smoke constituents compared to conventional cigarettes, the use of AMP in the paper of an EHC leads to further distinct reductions. In the study presented here, in vitro assays were used as quantitative tools to investigate toxicity-related mechanisms.

Exposure to airborne contaminants is significantly associated with human health risks, ranging from bronchial reactivity to morbidity and mortality due to acute intense or long term low level repeated exposures. However, the precise mechanisms that derive such effects are not always understood. Although inhalation studies are technologically complicated, correct hazard characterisation is essential for comparable risk assessment of inhaled materials. The aim of this study was to investigate the comparative in vitro cytotoxicity of selected gaseous contaminants in human lung cells. The cytotoxicity of nitrogen dioxide (NO(2)), sulphur dioxide (SO(2)) and ammonia (NH(3)) was investigated in A549- human pulmonary type II-like epithelial cell lines cultured on porous membranes in Snapwell inserts. A dynamic direct exposure method was established by utilizing the horizontal diffusion chamber system (Harvard Apparatus Inc, USA) for delivery of test atmospheres. Test atmospheres were generated using a dynamic direct dilution method and the concentration monitored by appropriate analytical methods. A diversified battery of in vitro assays including the MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays was implemented. Airborne IC(50) (50% inhibitory concentration) values were calculated based on the most sensitive assay for each test gas including NO(2) (IC(50)=11+/-3.54 ppm; NRU)>SO(2) (IC(50)=48+/-2.83 ppm; ATP)> and NH(3) (IC(50)=199+/-1.41 ppm; MTS). However, all in vitro assays revealed similar toxicity ranking for selected gaseous contaminants. Identical toxicity ranking was achieved using both in vitro and published in vivo data. This comparison suggests that results of in vitro methods are comparable to in vivo data and may provide greater sensitivity for respiratory toxicity studies of gaseous contaminants.

In order to elucidate the feasibility of predicting liver and kidney target-organ toxicity using in vitro cytotoxicity assay, cytotoxicity of selected chemicals, acetaminophen (AAP), mitomycin (MMC), cupric chloride (CuCl2), phenacetin, cadmium chloride (CdCl2) and aristolochic acid (AA), was studied using human hepatoma (Bel-7402) cells and human renal tubular epithelial (HK-2) cells. Cell viability and mitochondrial permeability transition (MPT) were assessed by the neutral red (NR) assay and laser scanning confocal microscope, respectively. The results of the NR assay indicated that cytotoxicity of hepatoxicants, AAP, MMC and CuCl2 in liver cells was higher than that in kidney cells. Cytotoxicitiy of nephrotoxicant, CdCl2 was lower in liver cells than that in kidney cells, but nephrotoxicant phenacetin and AA was higher cytotoxicity in liver cells than that in kidney cells. The cytotoxicity of AAP and phenacetin was strengthened in the presence of S9 mixture, indicating that they are metabolism-mediated cytotoxicants. All selected chemicals disrupted MPT in dose-dependent manner. Linear regression analysis revealed a good correlation between the IC50 values of cytotoxicity and the EC50 values of MPT in Bel-7402 cells and HK-2 cells (R2 = 0.987 and 0.823, respectively). Cytotoxicity assay in vitro using specific cells show good compatibility with target-organ toxicity in vivo. However, limitations of in vitro cytotoxicity assay are due to its incomplete process of ADME and the defect of predicting chronic toxicity effect after long-term exposure to a chemical.

This paper describes the in vitro cytotoxicity assessment of single walled carbon nanotubes (SWCNT) on A549 cells, a human lung cell line. Cellular viability was determined using the alamar blue (AB), neutral red (NR) and MTT assays, which evaluated metabolic, lysosomal and mitochondrial activity respectively. In addition, the total protein content of the cells was measured using the coomassie brilliant (CB) blue assay. Supernatants were also assayed for Adenylate Kinase (AK) release and Interleukin 8 (IL-8) which indicated a loss of cell membrane integrity and an inflammation response respectively. To investigate the interactions between serum components in the test medium and the test materials, exposures were conducted both in serum containing (5%) and serum-free medium. Results from the cytotoxicity tests (AB, CB, MTT) revealed the SWCNT to have very low acute toxicity to the A549 cells as all but one of the reported 24h EC(50) values exceeded the top concentration tested (800 microg/ml). The SWCNT were found to interfere with a number of the dyes used in the cytotoxicity assessment and we are currently conducting a comprehensive spectroscopic study to further investigate these interactions. Of the multiple cytotoxicity assays used, the AB assay was found to be the most sensitive and reproducible. Transmission electron microscopy (TEM) studies confirmed that there was no intracellular localization of SWCNT in A549 cells following 24h exposure; however, increased numbers of surfactant storing lamellar bodies were observed in exposed cells.

Basal level enzyme activities and enzyme inducibility were compared for rat hepatocytes that were cultured in 96-well plates with three different extracellular matrix configurations: single layer (SL) collagen type I, SL Matrigel, and collagen/Matrigel (C/M) sandwich. Overall, C/M sandwich and SL Matrigel plates were both superior to SL collagen type I plates in maintaining enzyme activities and inducibility and C/M sandwich plates had higher induced activity for CYP3A enzymes than SL Matrigel plates did. Cytotoxicity of nine reference compounds to rat hepatocytes (C/M sandwich configuration), rat hepatoma H4IIE and mouse fibroblast Balb/c 3T3 (3T3) cells was evaluated in 96-well plates using neutral red uptake (for 3T3) and tetrazolium salt MTS assays (for H4IIE and rat hepatocytes). For compounds chlorpromazine, quinidine, trichlorfon, thiopental, and antipyrine, the absolute differences in cytotoxicity LogIC(50) values obtained from different cell types were relatively small and without an obvious trend. The DeltaLogIC(50) values between cultured hepatocytes and the cell lines were much larger for acetaminophen and cyclophosphamide (1.35 < or =/DeltaLogIC(50)/ < or = 3.40), and for clofibrate and thioacetamide (not cytotoxic in hepatocytes at their highest dose levels). These large differences were likely the result of metabolism of these compounds in rat hepatocytes. The relationship between in vitro cytotoxicity LogIC(50) values and in vivo mouse or rat oral acute LogLD(50) values showed that compared to the cell lines, cultured rat hepatocytes improved correlation for acetaminophen and cyclophosphamide. The potential benefit of conducting in vitro cytotoxicity screening using a combination of permanent cell lines and cultured hepatocytes would allow us to obtain mechanistic insight on bioactivation, as well as improve the predictability of metabolism-mediated toxicity.

The cytotoxicity of three biocidal agents frequently employed as active ingredients in phenolic-based disinfectants, were evaluated in three established fish cell lines (EPC, CHSE and RTG-2). Cell viability was assessed using two fluorescent indicator dyes, Alamar Blue™ for metabolism and neutral red for lysosomal activity. Total protein content was also quantified as a measure of cell detachment. In order to evaluate the sensitivity of the cell cultures, the results obtained were compared with toxicity data obtained from a previous study with the same three compounds and the in vivo lethality test with rainbow trout. Results from this study established that each of the three cell lines ranked the tested chemicals in the same order of toxicity as the in vivo test; however, the cell cultures were found to be an order of magnitude less sensitive than whole fish studies with the same compounds. The chemical sodium o-benzyl-p-chlorophenol was consistently ranked the most toxic of the tested compounds with each cell line and the endpoints employed. The rank order of toxicity was always sodium o-benzyl-p-chlorophenol > sodium p-tertiary amylphenol > sodium o-phenylphenol. The EPC cells were found to be the most sensitive cell line tested based on Alamar Blue™ IC50 data, and the Alamar Blue™ assay was consistently found to be the most sensitive endpoint of the three cytotoxicity assays employed.

A naphthopyranone dimer, named planifolin, was isolated from a methylene chloride extract of the capitula of Paepalanthus planifolius Koern. The molecule (C(31)H(26)O(10)) appeared to be made up of two monomeric portions, semi-vioxanthin and paepalantine (an isocoumarin), linked by an ether bond, and it may possess several kinds of biological activity that can be related to its polyphenolic structure. Short-term tests that detect genetic damage can afford the information needed to evaluate carcinogenic risks of chemicals to humans. The Ames test, recommended for testing the mutagenicity of chemical compounds with potential pharmacological application, was used in the present study. The mutagenic activity was evaluated in Salmonella typhimurium strains TA100, TA98, TA102 and TA97a and the cytotoxic effect in McCoy cells. The in vitro cytotoxicity of planifolin to McCoy cells, tested in microculture with neutral red, showed a significant cytotoxic index (CI(50)) of 12.83 microg/mL. Planifolin showed mutagenic activity for TA100, TA98 and TA97a. The results indicate that this new naphthopyranone dimer causes mutations by substitution and by addition and deletion of bases in the sequence of DNA. Moreover, its mutagenic potential was increased by metabolic activation.

When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.

In order to investigate if a protein free cytotoxicity assay could improve the prediction of human acute toxicity, the cytotoxicity of 40 MEIC reference chemicals was measured by the neutral red uptake inhibition after 24 h in protein free culture medium on rat hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Potassium cyanide, arsenic trioxide, mercuric chloride, hexachlorophene and pentachlorophenol were much more cytotoxic in PF medium, as was the case to a lower extent for 16 other chemicals. The cytotoxicity of 8 chemicals was only changed to a limited extent when tested in PF medium, suggesting that serum proteins do not strongly interact with their cytotoxicity. Eleven other chemicals were less cytotoxic in PF medium, maybe because of too poor physiological conditions. Although a large number of differences in cytotoxicity were observed in function of the medium used for the assay, a good correlation was observed between both series of data ( r 2 = 0.946). The correlation between the cytotoxicity in PF medium and the human acute toxicity is lower ( r 2 = 0.647) than that in complete medium ( r 2 = 0.746). The results show that further research is necessary in order to improve the in vitro/in vivo correlations by introducing protein-dependent considerations.

The aim was to compare the use of different cell-material contact test methods with two different biological systems (cell line and tooth slice cultures) for cytotoxicity assessment of dental materials. Cytotoxicity of composites polymerized with two halogen-based and two light-emitting diode (LED) light-curing units (LCUs) served as the basis for comparison. Disk shaped specimens (7 x 2 mm) were fabricated using the four light sources. Composites were tested using L-929 cell line using direct/indirect/extract tests in accordance to standard protocols. Cytotoxicity was assessed using neutral red uptake. Tooth slice organ cultures were also employed to test the dental materials using direct/indirect test methods. Histomorphometric cell counting of intact odontoblasts and pulp fibroblasts and the use of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were applied for cytotoxicity evaluation. Discrepancy in result presentation was observed in the different tests used with L-929. Sensitivity levels of the L-929 tests ranked as follows: extract test < direct contact test < indirect contact test. Tooth slice tests confirmed that L-929 direct contact test proved to be the most reliable test among the three. In conclusion, this study highlights the risk involved when relying on a single test method for cytotoxicity assessment. It would be advisable to test different culture models and then proceed using more clinically relevant biological system that stimulate the in vivo situation for confirmation.

The use of FEO as a remedy for control of primary dysmenorrhea increases concern about its potential teratogenicity due to its estrogen-like activity. Limb bud mesenchymal cells, when grown in high-density cultures, can be differentiated into a number of cell types including cartilage and muscle. These cells have been used extensively for in vitro studies of chondrogenesis. Therefore, we used limb bud cells and Alcian blue staining method that is specific for staining cartilage proteoglycan, to determine the teratogenic effect of FEO. Limb bud cells obtained from day 13 rat embryo were cultivated and exposed to various concentrations of FEO for 5 days at 37 degrees C and the number of differentiated foci were counted. Retinoic acid (90 microg/ml) was chosen as positive standard control. The differentiation was also evaluated using limb bud micromass culture using immunocytochemical techniques and BMP-4 antibody. The results showed that FEO at concentration as low as 0.93 mg/ml produced a significant reduction in the number of stained differentiated foci. However, this reduction was due to cell loss, determined by neutral red cell viability assay, rather than to be related to decrease in cell differentiation. These findings suggest that the FEO at the studied concentrations may have toxic effect on fetal cells, but there was no evidence of teratogenicity.

Copper, though essential, is highly toxic when present in excess, as in Wilson disease, a genetic disorder of hepatic copper metabolism. We hypothesized that mitochondria are a major target of copper-induced cytotoxicity in Wilson disease. We used the human hepatoma line Hep G2 to examine copper-mediated cytotoxicity and three different methods to assess organelle damage: MTT assay (mitochondria), neutral red (NR; lysosomes) and Trypan blue exclusion assay (TB; plasma membrane). For all assays, cells at approximately 60% confluence in microtitre plates were incubated with CuCl(2) (concentration range: 50-100-150-200 microM) for 24 or 48 h. Results were expressed as percent of untreated control. At 24 h, cytotoxicity as detected by NR assay was significantly higher at all concentrations of copper than for MTT or TB ( p

Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2-3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring approximately 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 microg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 microg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 microg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring