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Background A sensitive method to investigate cellular stress and cytotoxicity is based on measuring mitochondrial membrane potential. Recently, JC-10, was developed to measure mitochondrial membrane potential in vitro and used as an indicator for cytotoxicity. Yet, JC-10 has never been used in vivo (whole organism). In normal cells, JC-10 concentrates in the mitochondrial matrix, where it forms red fluorescent aggregates. However, in apoptotic/necrotic cells, JC-10 diffuses out of the mitochondria, changes to monomeric form, and stains cells in green. Here, we aimed to develop and optimize a JC-10 assay to measure cytotoxicity in zebrafish embryo. We also investigated the effectiveness of JC-10 assay by comparing it to common cytotoxicity assays. Methods Zebrafish embryos were exposed to a toxic surfactant AEO-7 at no observed effect concentration (6.4 μg/L), and then cytotoxicity was measured using (i) JC-10 mitochondrial assay, (ii) acridine orange (AO), (iii) TUNEL assay, and (iv) measuring the level of Hsp70 by western blotting. Results As compared to the negative control, embryos treated with NOEC of AEO-7 did not show significant cytotoxicity when assessed by AO, TUNEL or western blotting. However, when JC-10 was used under the same experimental conditions, a significant increase of green:red fluorescent ratio signal was detected in the AEO-7 treated embryos, indicating mitochondrial damage and cellular cytotoxicity. Noteworthy, the observed green: red ratio increase was dose dependent, suggesting specificity of the JC-10 assay. Conclusion JC-10 is a sensitive in vivo method, thus, can be used as surrogate assay to measure cytotoxicity in whole zebrafish embryos.

Mono-2-ethyhexyl phthalate (MEHP), an environmental xenoestrogen, is widely used in the production of polyvinyl chloride materials and can be easily accumulated in human body. MBP is the active monoester metabolite of di butyl phthalate that is widely used as plasticizer in many products such as plastic toys, food packaging, personal care products, as well as an additive in lubricants, eliminating foams, and lotions. The presented in-vitro cytotoxicity study focused on time-dependent and combinatory exposure scenarios. We chose these phthalates because they are posed a considerable interest because of their contribution to insulin resistance, type-2 diabetes and obesity. All experiments performed in INS-1 pancreatic beta cells show moderate cytotoxicity with a time-dependent increase in effectiveness. INS-1 cells were treated with 0.001, 0.01, 0.1, 1, or 10-μM MEHP and MBP for 24, 48, and 72 h. Our results showed that cell viability was decreased and total oxidant levels were increased. Also, mRNA expression levels with asscociated beta cells were measured and for MBP dose groups, all mRNA expression levels were decreased. In conclusion, these findings suggest that, MEHP and MBP are have a negative and distruptor role on pancreatic beta cells and it will be linked with insulin resistance and type 2 diabetes.

The titanium dioxide nanoparticles (NPs) have been applied to biomedical, pharmaceutical, and food additive fields. However, the effect on health and the environment are conflicting; thus, it has been reviewing several times. In this context, establishing standard robust protocols for detecting cytotoxicity and genotoxicity of nanomaterials became essential for nanotechnology development. The cell type and the intrinsic characteristics of titanium dioxide NPs can influence nanotoxicity. In this work, the cyto- and genotoxicity effects of standard reference material titanium dioxide NPs in primary bovine fibroblasts and immortalized Chinese hamster ovary epithelial (CHO) cells were determined and compared for the first time. Titanium dioxide NPs exposure revealed no cytotoxicity for primary bovine fibroblasts, while only higher concentrations tested (10 μg/ml) induce genotoxic effects in this cell model. In contrast, the lower concentrations of the titanium dioxide NPs cause the cyto- and genotoxic effects in CHO cells. Therefore, our finding indicates that the CHO line was more sensitive toward the effects of titanium dioxide NPs than the primary bovine fibroblast, which should be valuable for their environmental risk assessment.

The cytotoxicity and DNA damage of titanium dioxide and zinc oxide nanoparticles (TiO2 and ZnO NPs) have been studied in a human lung carcinoma cell line (A549) after 24 h exposure. TiO2 and ZnO NPs had mean diameters of 12.9 ± 2.8 and 24.1 ± 8.0 nm, respectively. ZnO NPs reduced cell viability from 250 μg/mL, increasing reactive oxygen species (ROS) and decreased GSH/GSSG ratio. The comet assay detected DNA damage from 50 μg/mL. TiO2 NPs induced cytotoxicity and DNA damage from 50 to 100 μg/mL, respectively, along with a decrease of the GSH/GSSG ratio. Both particles were found inside the cells, within membrane-bound vesicles. The internalization mechanism is promoted partially by caveolae-mediated endocytosis and, in the case of TiO2 NPs, also by macropinocytosis.

Manganese (Mn) is an essential micronutrient. However, it is well established that Mn overexposure causes nervous system diseases. In contrast, there are few reports on the effects of Mn exposure on glomerular endothelium. In the present study, the potential effects of Mn exposure on glomerular endothelium were evaluated. Sprague Dawley rats were used as a model of Mn overexposure by intraperitoneal injection of MnCl2·H2O at 25 mg/kg body weight. Mn exposure decreased expression of vascular endothelial-cadherin, a key component of adherens junctions, and increased exudate from glomeruli in Sprague Dawley rats. Human renal glomerular endothelial cells were cultured with different concentration of Mn. Exposure to 0.2 mM Mn increased permeability of human renal glomerular endothelial cell monolayers and decreased vascular endothelial-cadherin expression without inducing cytotoxicity. In addition, Mn exposure increased phosphorylation of mothers against decapentaplegic homolog 2/3 and upregulated expression of zinc finger protein SNAI1, a negative transcriptional regulator of vascular endothelial-cadherin. Our data suggest Mn exposure may contribute to development of glomerular diseases by inducing permeability of glomerular endothelium.

Fine particulate matter (PM2.5) in the ambient atmosphere is strongly associated with detrimental health effects. However, these particles from various sources and regions are unlikely equally toxic. While animal studies are impractical for high-throughput toxicity testing, appropriate in vitro models are urgently needed. Co-culture of A549 and THP-1 macrophages grown at air–liquid interface (ALI) or under submerged conditions was exposed to same concentrations of ambient PM2.5 to provide accurate comparisons between culture methods. Following 24-h incubation with PM2.5 collected in Harbin in China, biological endpoints being investigated include cytotoxicity, reactive oxygen species (ROS) levels and pro-inflammatory mediators. The co-culture grown under submerged condition demonstrated a significant increase in ROS levels and all tested pro-inflammatory indicators [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] in mRNA expression and released protein levels. Similar but a declining response trend was observed using the same PM2.5 incubation after grown at ALI. We further observed a significant increase of PM2.5-induced phosphorylation of p38 MAPK and activation of NF-κB p65 in a dose-dependent trend for co-cultures grown under submerged condition. These results provide important implications that culture conditions (ALI versus submerged) can induce different extents of biological responses to ambient PM2.5; the co-culture grown at ALI is less likely to produce false-positive results than submerged culture. Hence, culture conditions should be discussed when comparing in vitro methods used for high-throughput PM2.5 toxicity assessment in future.

Hydrogels of TEMPO-oxidized nanocellulose were stabilized for dry-jet wet spinning using a shell of cellulose dissolved in 1,5-diazabicyclo[4.3.0]non-5-enium propionate ([DBNH][CO2Et]), a protic ionic liquid (PIL). Coagulation in an acidic water bath resulted in continuous core-shell filaments (CSFs) that were tough and flexible with an average dry (and wet) toughness of similar to 11 (2) MJ.m(-3) and elongation of similar to 9 (14) %. The CSF morphology, chemical composition, thermal stability, crystallinity, and bacterial activity were assessed using scanning electron microscopy with energy-dispersive X-ray spectroscopy, liquid-state nuclear magnetic resonance, Fourier transform infrared spectroscopy, thermogravimetric analysis, pyrolysis gas chromatography-mass spectrometry, wide-angle X-ray scattering, and bacterial cell culturing, respectively. The coaxial wet spinning yields PIL-free systems carrying on the surface the cellulose II polymorph, which not only enhances the toughness of the filaments but facilities their functionalization.

Ethnopharmacobotanical information reports that Parkinsonia aculeata infusion is used to control diabetes-related complications and dyslipidemia. However, few studies are reported on the safe use of this species. The aim of this study is to evaluate the acute toxicity, embryotoxicity and cytotoxicity of a polar fraction obtained from hydroethanolic extract of P. aculeata (PfrHEPA). For the acute toxicity test, we considered the Up and Down method which the guidelines are described by the Organization for Economic Cooperation and Development (OECD N°425). The animals were treated with PfrHEPA (2000 mg/kg) or with distilled water (10 ml/kg) by gavage and observed from Day 1 to14. For embryotoxicity assay, zebrafish embryos were exposed to PfrHEPA (100 mg/L) and toxicity parameters were observed during four consecutive days. The cytotoxicity of PfrHEPA (5, 10, 25, 50, 75 and 100 μg/ml, respectively) was performed on normal cell lines (mesenchymal stem cells, African green monkey renal cells and mouse pre-adipocytes 3 T3-L1 using the MTT salt reduction assay. In the acute toxicity test, no mortality was observed in mice treated with PfrHEPA (2000 mg/kg), as well as behavioral changes, histopathological abnormalities and hematological and biochemical variables. In the embryotoxicity test, no abnormal changes related to the toxicological parameters were observed in the period of 96 h. Regarding the cytotoxicity assay, PfrHEPA showed no cytotoxic effect on the normal cell lines tested, with an IC50 value > 100 μg/ml. These results suggest the safe use of P. aculeata, however, more trials are needed for PfrHEPA to be presented as new safe therapeutic proposal for the control of metabolic disorders.

The aim of the current study was to investigate the effect of well-characterized TiO2 nanoparticles on DNA methylation of peripheral blood mononuclear cells (PBMCs) in vitro. Maximum non-toxic concentration of nanoparticles for PBMCs was determined by MTT assay. The effect of TiO2 nanoparticles at concentrations of 25–100 μg/ml on DNA methylation of PBMCs was investigated by measuring the %5-mC alterations through an ELISA assay. The physicochemical analysis showed that the TiO2 nanoparticles were crystalline, pure and in the anatase phase. Peaks related to Ti-O tensile vibrations were observed in the range of 1510 cm−1. The size of nanoparticles was in the range of 39–74 nm with an average hydrodynamic diameter of 43.82 nm. According to the results of the MTT test, 100 μg/ml was found to be maximum non-toxic concentration. The %5-mC in treated PBMCs revealed that TiO2 nanoparticles could lead to DNA hypomethylation in PBMCs. The %5-mC difference compared with the negative control was found to be 2.07 ± 1.02% (P = 0.03). The difference of %5-mC between the 25 and 100 μg/ml concentration of nanoparticles was statistically significant (P = 0.02). The results of the current study show that the TiO2 nanoparticles cause DNA hypomethylation in PBMCs in a dose-response manner. Therefore, it is recommended to evaluate the effects of cytotoxicity and epigenotoxicity of commonly used nanoparticles before their use.

This study was aimed to evaluate toxicity in repeated doses for 28 days, reproductive toxicity and cytotoxicity of a polar fraction obtained from the hydroethanolic extract of Parkinsonia aculeata (PfrHEPA) in experimental models. To perform the toxicity test in repeated doses for 28 days, male and female Wistar rats were treated via orogastric for 28 days with PfrHEPA (35, 70 or 140 mg/kg) according to the guidelines established by the Organisation for Economic Co-operation and Development (OECD) number 407 (1995). For assessment, the impact of PfrHEPA on the reproductive output various parameters were measured, including maternal weight, no. of pregnant females, female fertility index (%), gestation lengthtime, implantation sites, litter size and placental index of test animals. The cytotoxicity of PfrHEPA was performed on the tumor lines NCI-H292 (human lung carcinoma), HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer). In the repeated dose toxicity test for 28 days, no mortality was observed in the male and female rats treated with PfrHEPA as well as morphological changes and biochemical and hematological parameters. In the reproductive toxicity test, no abnormalities were observed related to the toxicological parameters in both mothers and offspring. Regarding the cytotoxicity assay, the PfrHEPA fraction did not demonstrate significant cytotoxic effect on the cell lines analyzed. The present results suggest the use of PfrHEPA is safe and well tolerated in rats. Further studies are planned to identify and purify the active compounds for subsequent in vivo evaluation.

The wide uses of graphene oxide (GO) lead to the contact of GO with vascular systems, so it is necessary to investigate the toxicological effects of GO to endothelial cells. Recently, we reported that GO of small lateral size (

Pomegranate (Punica granatum L.) is a fruit used extensively in traditional medicine by ancient and modern cultures. Different parts of the tree and fruit, such as leaf, peel, pericarp, aril, seed, and juice contain considerable amounts of phenolic compounds with high antioxidant activities. To improve its storability, pomegranate juice was microencapsulated by spray drying. The present study evaluated microencapsulated pomegranate juice (MPJ) for toxic effects in Wistar rats and CD-1 mice to determine if MPJ can be considered safe for human consumption and used as a nutraceutical. No deaths or deleterious effects occurred when high doses of 5000 mg/kg were orally administered in rats for 14 days, indicating an absence of subacute toxicity. Similarly, 3000 mg/kg MPJ administered to CD-1 mice for 90 days did not show subchronic toxicity. In fact, MPJ resulted in lowered weight gain in both rats and mice. Cytotoxic and microbiological analyses of MPJ were also performed. MPJ did not cause any cytotoxicity in epithelial cell culture as tested using the Alamar blue assay. Additionally, histopathological analysis of kidney and liver corroborated the absence of toxicity in CD-1 mice. The microbial load of the MPJ was low, and no pathogenic bacteria were present. In conclusion, the results reported here show that high doses of MPJ are apparently innocuous in rats and mice for the 14 and 90 days investigated, respectively. Although preliminary, our results suggest that MPJ may be safe to ingest and may even have beneficial effects in reducing weight gain.

The cytotoxicities of silica (SiO2s) particles against cancers are still controversial. In this study, the purchased submicron silica particles (SM-SiO2s) were identified by transmission electron microscopy and energy dispersive spectrometer, and it showed potent cytotoxicities on hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC) and breast cancer (BC), which ranked the top in the incidence among the tumor types. Through the microarray assay on long noncoding RNAs (lncRNAs) from the SM-SiO2s-treated HCC, NSCLC and BC cells, followed by Venn analysis, we found that a series of lncRNAs were significantly regulated by SM-SiO2s, among of which XLOC_001659 was mostly decreased. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay confirmed that XLOC_001659 could be decreased in all the SM-SiO2s-treated HCC, NSCLC and BC cells, coupled to inhibited cell proliferation. Further, XLOC_001659 was recognized as a miR-98-5p sponge and therefore modulates the “pro-inflammatory tumor promoter” MAP3K2 expressions. The XLOC_001659/miR-98-5p/MAP3K2 axis uniformly mediated the regulation of SM-SiO2s on proliferation of HCC, NSCLC and BC cells. Further clinical experiments demonstrated that XLOC_001659 was negatively correlated with miR-98-5p level and positively correlated with MAP3K2 level, and XLOC_001659/miR-98-5p/MAP3K2 axis was significantly associated with progressions and prognosis in HCC, NSCLC and BC patients. These results provide a new clue for the anti-tumor mechanism of SM-SiO2s and a new way for drug development by using SM-SiO2s.

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca2+ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca2+ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca2+ concentration ([Ca2+]i) was measured by the Ca2+-sensitive fluorescent dye fura-2. Theophylline (25–125 μM) induced [Ca2+]i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished theophylline-induced [Ca2+]i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca2+]i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca2+]i rises. In Ca2+-containing medium, modulators of store-operated Ca2+ channels inhibited 30% of the [Ca2+]i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca2+-associated cytotoxicity and induced Ca2+ entry through PLC-independent Ca2+ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca2+ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity.

The specific cytotoxic effect of nanoparticles on tumor cells may be used in future antitumor clinical applications. Silver nanoparticles (AgNPs) have been reported to have potent cytotoxic effect, but the mechanism is unclear. Here, AgNPs were synthesized, and the particle average size was 63.1 ± 8.3 nm and showed a nearly circular shape, which were determined by transmission electron microscopy and field emission scanning electron microscopy. The selected area electron diffraction patterns showed that the nanoparticles were crystalline. The energy-dispersive X-ray spectrum proved that silver is the main component of nanoparticles. The AgNPs showed potent cytotoxicity in breast cancer cells, no matter whether they were tamoxifen sensitive or resistant. Next, we found that a long noncoding RNA, XLOC_006390, was decreased in AgNPs-treated breast cancer cells, coupled to inhibited cell proliferation, altered cell cycle and apoptotic phenotype. Downstream of AgNPs, XLOC_006390 was recognized to target miR-338-3p and modulate the SOX4 expression. This signaling pathway also mediates the AgNPs function of sensitizing tamoxifen-resistant breast cancer cells to tamoxifen. These results provide a new clue for the antitumor mechanism of AgNPs, and a new way for drug development by using AgNPs.

A novel simple and readily synthesized turn-on fluorescent probe 4-(benzo[d]thiazol-2-yl)-1,3-phenylene bis(2-chloroacetate) (BPBC) for cysteine (Cys) was reported. This probe was designed based on an excited-state intramolecular proton transfer (ESIPT) dye: benzothiazole, and two chloroacetate groups present in benzothiazole as the reaction sites for Cys. It shows high selectivity and sensitivity for Cys over other amino acids including the similar structured homocysteine (Hcy) and glutathione (GSH). In addition, probe BPBC was successfully applied to bioimage intracellular Cys in living cells with low cytotoxicity. More importantly, a paper test strip system was developed with probe BPBC for Cys detection conveniently.

Significance: Worldwide, ∼460 million people suffer from disabling hearing impairment. Many of these patients are still not sufficiently supplied with currently available auditory technologies. Optical stimulation of the hearing organ offers a promising alternative for a new generation of auditory prostheses. Aim: To assess the biocompatibility margins of our laser pulse amplitude strategy in vitro, we designed a protocol and present the effects on normal human dermal fibroblasts, human chondrocytes, and human osteoblasts. Approach: Laser pulses of 532 nm were applied over 120 s using our laser pulse amplitude modulation strategy. We then assessed cell viability and cytotoxicity through fluorescence staining and quantitative polymerase chain reaction-analysis regarding 84 key player-genes for cytotoxicity and stress response. Results: The first in vitro biocompatibility margins for our stimulation parameters applied to cells of the peripheral hearing organ were between 200 and 223 mW (3348 J/cm2). After irradiation with a subphototoxic laser power of 199 mW (2988 J/cm2), only the fibroblasts showed a significant upregulation of GADD45G. Conclusion: Further studies are underway to optimize parameters for the optoacoustic stimulation of the auditory system. Our protocol and results on laser–tissue interactions can be useful for translational laser applications in various other irradiated biological tissues.

Significance: Currently, various scaffolds with immobilized cells are widely used in tissue engineering and regenerative medicine. However, the physiological activity and cell viability in such constructs might be impaired due to a lack of oxygen and nutrients. Photobiomodulation (PBM) is a promising method of preconditioning cells to increase their metabolic activity and to activate proliferation or differentiation. Aim: Investigation of the potential of PBM for stimulation of cell activities in hydrogels. Approach: Mesenchymal stromal cells (MSCs) isolated from human gingival mucosa were encapsulated in modified fibrin hydrogels with different thicknesses and concentrations. Constructs with cells were subjected to a single-time exposure to red (630 nm) and near-infrared (IR) (840 nm) low-intensity irradiation. After 3 days of cultivation, the viability and physiological activity of the cells were analyzed using confocal microscopy and a set of classical tests for cytotoxicity. Results: The cell viability in fibrin hydrogels depended both on the thickness of the hydrogels and the concentration of gel-forming proteins. The PBM was able to improve cell viability in hydrogels. The most pronounced effect was achieved with near-IR irradiation at the 840-nm wavelength. Conclusions: PBM using near-IR light can be applied for stimulation of MSCs metabolism and proliferation in hydrogel-based constructs with thicknesses up to 3 mm.

Cytotoxic T lymphocytes (CTLs) play a key role in adoptive cell therapy (ACT) by destroying tumor cells. Although some mechanisms of CTLs killing tumor cells have already been revealed, the precise dynamic information of CTLs’ interaction with tumor cells is still not known. Here, we used confocal microscopy to visualize the whole process of how CTLs kill tumor cells in vitro. According to imaging data, CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or more tumor cells, and the average time for CTLs destroying one or more tumor cells in vitro is dozens of minutes only. Our study displayed the temporal events of CTLs’ interaction with tumor cells at the beginning up to the point of killing them. Furthermore, the imaging data presented strong cytotoxicity of CTLs toward the specific tumor cells. These results could help us to well understand the mechanism of CTLs’ elimination of tumor cells and improve the efficacy of ACT in cancer immunotherapy.

Ricin is a highly-toxic compound derived from castor plant beans. Several incidents involving contamination of residences and buildings due to ricin production or dissemination have occurred in recent years. The goal of this study was to determine whether ricin bioactivity could be attenuated in reasonable time via simple modifications of the indoor environment. Attenuation was assessed on six different materials as a function of temperature, relative humidity (RH), and contact time, using both a pure and crude preparation of the toxin. Ricin bioactivity was quantified via a cytotoxicity assay, and attenuation determined as the difference in ricin recovered from test and positive controls. The results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent and results more variable. We found no significant attenuation in crude ricin after two weeks at typical indoor environmental conditions, except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40°C for 5 days or 50°C for 2–3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40°C after two weeks.

WOS: 000313019700012, PubMed ID: 22271281, The purposes of this study were to investigate the biocompatibility of two different paper patches (carbon and cigarette papers) and compare the adhesion and proliferation features of L929 fibroblast cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT Test) test and scanning electron microscopy (SEM). In this study, time-dependent cytotoxic effects of cigarette and carbon papers used in repairing small traumatic TM perforations were investigated in vitro by using MTT test. And also adhesion and spreading of cells over disk surface were observed by SEM. Cytotoxicity test carried out by MTT analysis on leakage products collected from two types of paper patches at the end of 24 and 48 h revealed no cytotoxicity (P > 0.05). In SEM studies, it was observed that cells started to proliferate over disk surface as a result of 48-h incubation, and SEM revealed that the cell proliferation over cigarette paper was more compared to the one over carbon paper. We believe that this is the first study where biocompatibility and adhesion features of carbon and cigarette paper have been studied by using L929 fibroblast cell culture. As a result, biocompatibility of cigarette paper and also whether cigarette paper was superior to carbon paper in cell attachment and biocompatibility were studied. It was found, by MTT test and SEM test, that cigarette paper had a higher biocompatibility and cell attachment, and thus cigarette paper should be the patch to be preferred in cases where TM perforations are repaired by paper-patch method.

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.

Objective: To evaluate the toxicity of extracts from disposable chopsticks, toothpicks, and paper cups on L-929 cells. Methods: We followed national standards to prepare the extracts from disposable chopsticks, toothpicks, and paper cups used for the cell culture media, and the morphology of L-929 cells was observed with an optical microscope. The loss rate for adherent cells was evaluated with the trypan blue exclusion method, and cell proliferation was determined using the WST-1 assay. Results: Compared with the control group, the cells cultured in media containing the extracts showed signs of apoptosis and necrosis after culturing for 4 or 7 days, and the loss rate for adherent cells was significantly increased (P < 0.05). An obvious decrease in cell viability was also observed (P < 0.05). Conclusion: The extracts from disposable chopsticks, toothpicks, and paper cups can affect the growth and proliferation of L-929 cells and are potentially toxic to humans.

Because of the lack of information about the possible transfer of toxic compounds from papers and boards to food, the overall cytotoxicity induced by six papers and 15 boards was investigated from water extracts prepared according to the European prestandard. Cytotoxicity measurements were based on RNA synthesis rate of human HeLa S3 cells. The tested virgin and recycled papers and boards were differentiated and classified according to their cytotoxicological quality, which ranged from absence of any cytotoxic effect to severe inhibition of RNA synthesis rate. The cytotoxicity level also varied according to the total amount of compounds detected by gas chromatography. No correlation was found between cytotoxicity and endotoxins contained in the samples. No significant difference in cytotoxicity was observed between the papers and boards produced from virgin fibres and from recycled fibres. Moreover, the products obtained from chemical pulp showed lower cytotoxicity than the products based on mechanical pulp. More generally, the cytotoxicological approach is promising for monitoring paper/board treatment-induced problems. Further work is required to assess a modified standard procedure for the preparation of water extracts specifically adapted to paper/board.

International audience; In vitro toxicological tests has have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. An EU 5th framework program project “BIOSAFEPAPER – Application of bioassays for safety assessment of paper and board for food contact” specially focused on the application of biotests to paper and board. The project included both chemical characterization and toxicological testing of a representative number of paper and board extracts prepared according to the proposed end use (wet, fatty and dry food contact). Among the concerns addressed in the project wereraised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in paper and boardchemically complex materials such as paper and board, either as constituents or contaminants.. In order to answer these questions, a series of experiments were performed to assess the role of, natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzoa(a)pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates , were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments the extracts did not affect the toxicity of tested chemicals, nor was there any significant metabolic intereference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, biotesting can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by the chemical analysis alone.

International audience; Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Dichloromethane extracts of four diverse hazardous wastes (coke plant, herbicide manufacturing, pulp and paper, and oil refining) were evaluated for mutagenicity in strains TA98 and TA100 of Salmonella. These extracts also were tested for biological activity in short-term mammalian cell assays, including mutagenicity in L5178Y/TK +/- mouse lymphoma cells, chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells, morphological transformation in BALB/c-3T3 cells, and teratogenic potential in mouse limb bud cells. The mammalian cell assays were performed using limited protocols that consisted of a preliminary testing of the extracts for cytotoxicity in CHO cells in order to estimate the appropriate dose range for the other assays. These assays were then performed once with only a few doses of extract; all but the mouse limb bud assay were performed in the presence of metabolic activation. Although all four of the wastes were presumptively positive for either mutation or cytogenetic effects, none of the wastes transformed BALB/c-3T3 cells. Further studies are needed to establish which mammalian cell assays, if any, might be useful complements to the Salmonella assay for the purpose of screening hazardous wastes.

Summary. The biocompatibility of absorbent paper points was evaluated by a haemolysis test and a tissue culture agarose overlay test. One type of paper points produced marked haemolytic activity and a cytotoxic response in the overlay test. The other types tested showed minimal haemolytic activity and no cytotoxicity. Chemical analysis of the cytotoxic paper points revealed that they contained formaldehyde.