Mutagenic, cytotoxic, and genotoxic properties of tobacco smoke produced by cigarillos available on the Canadian market

Cigarillos (aka little cigars) have been increasing in popularity unlike cigarettes; but relatively little is known about the toxicology of the mainstream smoke (MSS) from such products. Therefore, the objective of this work was to compare the toxicological properties of the MSS (Health Canada Intensive smoking conditions) from a range of cigarillo products with the toxicological properties of MSS of cigarettes. Three in vitro assays were used to evaluate the toxicities of the MSS total particulate matter (TPM): (1) mutagenicity using Ames assay with Salmonella strains TA98 and TA100 with S9 metabolic activation (+S9); (2) cytotoxicity using the Neutral Red Uptake (NRU) assay with CHO (Chinese Hamster Ovary) cells; and (3) genotoxicity using the micronucleus assay with CHO cells and short-term exposures (3-h ± S9). The Ames assay (TA100 + S9) and the NRU assay were also applied to the gas/vapour phase of the MSS that passed through the Cambridge pad. On a per-milligram-nicotine basis, the preferred way of comparing toxicities of different types of tobacco products, the MSS from cigarillos was not less toxic, and in some cases more toxic (TPM fraction TA98 + S9, NRU), than the MSS from cigarettes. Thus, our findings support our prior work on smoke mutagenicity that showed MSS from cigarillos was not less toxic than MSS from cigarettes.     

Effect of cigarette filters on the chemical composition and in vitro biological activity of cigarette mainstream smoke

The objective of this study was to evaluate the effects of cigarette filters on the chemical composition and toxicity of cigarette mainstream smoke. In this work, we used three types of cigarettes, including non-filter 2R4F cigarettes, cellulose acetate (CA)-filter 2R4F cigarettes, and carbon dual-filter 2R4F cigarettes. The cytotoxicity of TPM obtained from the filter cigarettes was not different from that of the non-filter cigarettes on an equal TPM basis. However, the EC₅₀ vlaue of GVP from carbon-filter cigarettes were 40.9 puffs/L, thereby indicating the cytotoxicity of these cigarettes was approximately 37% and 21% lower than non-filter and CA-filter cigarettes, respectively. The cytotoxicity of GVP was correlated with carbonyl components. The mutagenicity of TPM obtained from non-filter cigarettes, calculated on an equal TPM basis, was up to 30–40% lower than that of the filter cigarettes. When calculated on a per cigarette basis, the mutagenicity of CA or carbon-filter cigarettes was found to be 35% lower than that of the non-filter cigarettes. The results of chemical composition analyses revealed that the observed increase in aromatic amine compound yields on an equal TPM basis in filter cigarettes may be related with the mutagenic activity determined in Ames assays.     

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter

A novel carbon filter has been developed which primarily reducesthe amount of certain vapor phase constituents of tobacco smokewith greater efficiency than the charcoal filters of cigarettescurrently in the market In vitro indicators of genotoxic andcytotoxic potential were used to compare the cigarette smokecondensate (particulate phase) or whole cigarette smoke (vaporphase and particulate phase) from cigarettes containing thenovel carbon filter with smoke condensate or whole smoke fromcommercial or prototype cigarettes not containing the novelcarbon filter. Ames bacterial mutagenicity, sister chromatidexchange (SCE) in Chinese hamster ovary (CHO) cells, and neutralred cytotoxicity assays in CHO cells were utilized to assessthe genotoxic and cytotoxic potential of the cigarette smokecondensates. SCE and neutral red cytotoxicity assays were utilizedto assess the genotoxic and cytotoxic potential of the wholesmoke. As expected, the novel carbon filter did not significantlyaffect the genotoxic or cytotoxic activity of the smoke condensate,although we did observe that the use of low-nitrogen tobaccoreduced the mutagenicity of the condensate in Salmonella typhimuriumstrain TA98. However, the whole smoke from cigarettes containingthe novel carbon filter demonstrated significant reductionsin genotoxic and cytotoxic potential compared to cigaretteswithout the novel carbon filter. The toxicity of the smoke wascorrelated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCEinduction) to the aggregate mass of several vapor phase components(acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene,ammonia, NO_x, HCN, benzene, isoprene, and formaldehyde) in thesmoke of the cigarettes utilized in this study. In conclusion,this novel carbon filter, which significantly reduced the amountof carbonyls and other volatiles in mainstream cigarette smoke,resulted in significant reductions in the genotoxic and cytotoxicactivity of the smoke as measured by these assays.     

Effects of smoking regimens and test material format on the cytotoxicity of mainstream cigarette smoke

The purpose of this study was to evaluate the effects of test material format and smoking regimens on comparative toxicity testing of cigarette smoke. Total particulate matter (TPM) or whole smoke (WS) generated from three test cigarettes under International Organization for Standardization (ISO) or Health Canada Intensive (HCI) regimens were assessed for cytotoxicity using the neutral red uptake (NRU) cytotoxicity assay. Under both ISO and HCI regimens, the relative differences of cytotoxicity among the test cigarettes indicated by the EC50 values in WS were significantly higher than those in TPM. For TPM testing, cytotoxicity was decreased going from ISO regimen to HCI regimen, consistent with the reported reductions of toxicant output on a per unit of TPM basis under the HCI regimen. For WS, cytotoxicity increased for the two lower TPM cigarettes, and decreased for the higher TPM cigarette going from HCI regimen to ISO regimen. Results from this study demonstrated WS should be the preferable test material format for smoke toxicity testing whenever possible. Intensive smoking regimens, such as HCI, are less likely to underestimate smoke toxicant intakes by smokers, and should be included in the comparative toxicological testing strategy.     

Thirdhand tobacco smoke: procedures to evaluate cytotoxicity in cell cultures

The risks associated to tobacco smoking are not ceased with smoke extinction. Many toxic compounds remain in the environment after the cigarette is extinguished and accumulated in the air or on surfaces. However, little is known about the risks of this exposure. The aim of this study was to evaluate procedures to collect thirdhand smoke (THS) and prepare the samples to perform three in vitro toxicity tests. Cellulose papers and cotton wipes were used to impregnate with nicotine solution and smoke cigarette in a chamber or in smoker’s home. Samples were immersed in methanol or Dulbecco’s modified Eagle’s medium (DMEM) to expose Hep-2 cells. MTT, neutral red uptake (NRU) and trypan blue assays were performed. The concentration of nicotine in DMEM extract of THS in paper and cotton was similar to those in methanol extract (p?>?0.05). Alterations in the mitochondrial and lysosomal functions were found in both paper and cotton samples; however, the cytotoxic effect was not always observed. There was a decrease of 21–31% in MTT assay and 38–56% in NRU assay (p?<?0.003). There was a dose-response relationship between the amount of cigarettes and lysosomal viability; the correlation was higher for cotton samples (r?=??0.843, p?<?0.001). As a dose-response relationship was found only in NRU assay, this test may be a more suitable choice rather than the MTT assay. Paper and wipe sampling can be reliable markers of tobacco smoke contamination. Moreover, these materials, if properly prepared, can be used as substrate providers to perform cellular assays.     

E‐cigarettes: voltage‐ and concentration‐dependent loss in human lung adenocarcinoma viability

E‐cigarettes are used by millions of people despite the fact that the harmful effect of aerosol emitted from these products to the human organism is still not clear. In this paper, toxicity of vapor generated using different solutions and battery output voltage on A549 cells viability is presented. The obtained EC₅₀ values for commercially available propylene glycol/glycerol solution 1:1 e‐liquids based on 3.2 V (0.127%), 4.0 V (0.112%) and 4.8 V (0.038%) were about 1.5–4.5 times higher than in tobacco smoke (0.0086%). Furthermore, it was shown that the increase of battery output voltage decreased A549 cell viability. In addition, commercially available extracts were more cytotoxic than laboratory made extracts. Owing to the expansiveness of e‐cigarettes, it is very important to estimate their impact on public health. Our results not only confirm less cytotoxicity of e‐liquid aerosol than cigarette smoke, but also demonstrate that solutions used in e‐liquids and, for the first time, battery output voltage have a significant impact on cytotoxicity of e‐cigarette vapor. Thus, the results of this study are very important for the current and future legal regulations on e‐cigarettes.     

Use of in vitro assays to assess the potential cytotoxic,genotoxic and antigenotoxic effects of vanillic and cinnamic acid

Vanillic acid (VA) found in vanilla and cinnamic acid (CA) the precursor of flavonoids and found in cinnamon oil, are natural plant phenolic acids which are secondary aromatic plant products suggested to possess many physiological and pharmacological functions. In vitro and in vivo experiments have shown that phenolic acids exhibit powerful effects on biological responses by scavenging free radicals and eliciting antioxidant capacity. In the present study, we investigated the antioxidant capacity of VA and CA by the trolox equivalent antioxidant capacity (TEAC) assay, cytotoxicity by neutral red uptake (NRU) assay in Chinese Hamster Ovary (CHO) cells and also the genotoxic and antigenotoxic effects of these phenolic acids using the cytokinesis-blocked micronucleus (CBMN) and the alkaline comet assays in human peripheral blood lymphocytes. At all tested concentrations, VA (0.17–67.2?μg/ml) showed antioxidant activity but CA (0.15–59.2?μg/ml) did not show antioxidant activity against 2,2-azino-bis (3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS). VA (0.84, 4.2, 8.4, 16.8, 84 and 168?μg/ml) and CA (0.74, 3.7, 7.4, 14.8, 74, 148?μg/ml) did not have cytotoxic and genotoxic effects alone at the studied concentrations as compared with the controls. Both VA and CA seem to decrease DNA damage induced by H₂O₂ in human lymphocytes.     

The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields

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.     

Evaluation of cytotoxicity of different tobacco product preparations

Acute exposure to cigarette smoke or its components triggers diverse cellular effects, including cytotoxicity. However, available data regarding the potential cytotoxic effects of smokeless tobacco (ST) extracts lack consensus. Here, we investigated the relative biological effects of 2S3 reference ST, and whether ST elicits differential cellular/molecular responses compared to combustible tobacco product preparations (TPPs) prepared from 3R4F cigarettes. Total particulate matter (TPM) and whole smoke conditioned medium (WS-CM) were employed as combustible TPPs, while the ST extract was used as non-combustible TPP. HL60, THP1 cells and human PBMCs were used to examine the effects of TPPs in short-term cell culture. Corresponding EC₅₀ values, normalized for nicotine content of the TPPs, suggest that combustible TPPs induced higher cytotoxicity as follows: WS-CM ? TPM ? ST extract > nicotine. While all three TPPs induced detectable levels of DNA damage and IL8 secretion, the combustible TPPs were significantly more potent than the ST preparation. The major PBMC subsets showed differential cytotoxicity to combustible TPPs as follows: CD4 > CD8 > monocytes > NK cells. These findings suggest that, relative cytotoxic and other cell biological effects of TPPs are dose-dependent, and that ST extract is the least cytotoxic TPP tested in this study.     

Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose–response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.     

Cytotoxicity of carbon nanotube variants: A comparative in vitro exposure study with A549 epithelial and J774 macrophage cells

Abstract

While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO₂ and SiO₂ reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs’ physico–chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes.     

Toxicity profiles of four metals and 17 xenobiotics in the human hepatoma cell line HepG2 and the protozoa Tetrahymena pyriformis—A comparison

We performed an interspecies comparison for the human hepatoma cell line HepG2 and the eukaryotic single cell organism Tetrahymena pyriformis (T. pyriformis) for 17 xenobiotics with diverse structures and four metals. The cytotoxicity was assessed by four different cell viability assays (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide reduction (MTT), neutral red uptake (NRU), resazurin dye (AlamarBlue), 5‐carboxyfluorescein diacetate acetoxymethyl ester (CFDA‐AM)) for the HepG2 and by cell count and MTT for T. pyriformis. For HepG2 cells, the results revealed interassay variations depending on the compound. The highest assay conformity was found for the metal Hg²⁺ and the fungicide prochloraz. The AlamarBlue assay was the most sensitive assay according to low‐effect concentrations. By contrast, the NRU assay was comprised of more frequent whole concentration response relationships and was more susceptible to EC₅₀. For T. pyriformis the EC₅₀ values of the two applied assays displayed a high conformity (R² = 0.97). Comparing the EC₅₀ values obtained by the MTT assay for the two cell models, a direct correlation was absent for the xenobiotics and only present for the metals (Cd²⁺, Cu²⁺, and Ni²⁺). Moreover, the protozoa T. pyriformis displayed a 20 times higher sensitivity than the cell line. The highest interspecies difference of three log degrees was obtained for the polycyclic aromatic hydrocarbon fluoranthene. In addition, a correlation of the EC₅₀ values and octanol‐water partition coefficient (log K_OW) of the xenobiotics was performed. No correlation was found for HepG2, and a weak one for T. pyriformis. Interestingly, the interspecies difference of logarithmized EC₅₀ correlated positive with the log K_OW (R² = 0.65). In conclusion, to obtain reliable evidence for human cytotoxicity, more than one viability/cytotoxicity assay had to be applied for cell lines. Second, the human hepatoma cell line was less affected by the organic compounds than the eukaryotic single‐cell organism and was also less dependent on the log K_OW of the xenobiotic. © 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 171–186, 2011.     

Design,synthesis, and evaluation of the anticancer activity of 2‐amino‐aryl‐7‐aryl‐benzoxazole compounds

A series of 2‐amino‐aryl‐7‐aryl‐benzoxazole derivatives have been designed, synthesized, and evaluated as anticancer agents. Fourteen of the compounds exhibited cytotoxic effects toward human A549 lung cancer cells. We found 12l was the most potent with an EC₅₀ of 0.4 μm , equivalent to the anticancer drug doxorubicin, but had low selectivity following cross screening in monkey kidney Vero cells. Eight of the most potent or most selective compounds were further profiled in additional cell lines (MCF7, NCI‐H187, and KB) to better understand their cytotoxic activity. Only compound 12l had a measurable EC₅₀ in a single cell line (3.3 μm in the KB cell line). Taken together, this data suggest the series as a whole display specific cytotoxicity toward A549 cells. Cheminformatics searches pointed to JAK2 as a possible target. A subset of compounds assayed at this target showed IC₅₀s ranging from 10 to 0.08 μm ; however, no clear correlation between JAK2 potency and A549 cytotoxicity was observed.     

Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose–response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.     

Comparative assessment of three in vitro exposure methods for combustion toxicity

A comparative assessment of three approaches for the use of human cells in vitro to investigate combustion toxicity was conducted. These included one indirect and two direct (passive and dynamic) exposure methods. The indirect method used an impinger system in which culture medium was used to trap the toxicants, whilst the direct exposure involved the use of a Horizontal Harvard Navicyte Chamber at the air/liquid interface. The cytotoxic effects of thermal decomposition products were assessed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega) on a selection of human cells including: HepG2, A549 and skin fibroblasts. A small scale laboratory fire test using a vertical tube furnace was designed for the generation of combustion products. Polymethyl methacrylate (PMMA) was selected as a model polymer to study the cytotoxic effects of combustion products. NOAEC (no observable adverse effect concentration), IC10 (10% inhibitory concentration), IC50 (50% inhibitory concentration) and TLC (total lethal concentration) values were determined from dose response curves. Assessment using the NRU (neutral red uptake) and ATP (adenosine triphosphate) assays on human lung derived cells (A549) was also undertaken. Comparison between in vitro cytotoxicity results against published toxicity data for PMMA combustion and predicted LC50 (50% lethal concentration) values calculated from identified compounds using GCMS (gas chromatography mass spectrometry) was determined. The results suggested that the indirect exposure method did not appear to simulate closely exposure via inhalation, whilst exposure at the air/liquid interface by using the dynamic method proved to be a more representative method of human inhalation. This exposure method may be a potential system for in vitro cytotoxicity testing in combustion toxicity.     

The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays

Particulate matter (PM) collected from mainstream tobacco smoke is a test article commonly used for in vitro genotoxicity and cytotoxicity testing of combustible tobacco products. However, little published data exists concerning the stability of PM. We completed a 2 year study to quantify the effect of PM storage at ?80 °C, on the genotoxicity and cytotoxicity of PM generated from 3R4F and M4A reference cigarettes. The Ames test, Micronucleus assay (MNvit), Mouse Lymphoma assay (MLA) and the Neutral Red Uptake assay (NRU) were used. The majority of M4A and 3R4F PMs were genotoxic and cytotoxic at the timepoints tested. Some minor but statistically significant differences were observed for stored versus freshly prepared PM, but the magnitude of changes were within the variability observed for repeat testing.     

Evaluation of cytotoxicity of different tobacco product preparations

Acute exposure to cigarette smoke or its components triggers diverse cellular effects, including cytotoxicity. However, available data regarding the potential cytotoxic effects of smokeless tobacco (ST) extracts lack consensus. Here, we investigated the relative biological effects of 2S3 reference ST, and whether ST elicits differential cellular/molecular responses compared to combustible tobacco product preparations (TPPs) prepared from 3R4F cigarettes. Total particulate matter (TPM) and whole smoke conditioned medium (WS-CM) were employed as combustible TPPs, while the ST extract was used as non-combustible TPP. HL60, THP1 cells and human PBMCs were used to examine the effects of TPPs in short-term cell culture. Corresponding EC₅₀ values, normalized for nicotine content of the TPPs, suggest that combustible TPPs induced higher cytotoxicity as follows: WS-CM ⩾ TPM ≫ ST extract > nicotine. While all three TPPs induced detectable levels of DNA damage and IL8 secretion, the combustible TPPs were significantly more potent than the ST preparation. The major PBMC subsets showed differential cytotoxicity to combustible TPPs as follows: CD4 > CD8 > monocytes > NK cells. These findings suggest that, relative cytotoxic and other cell biological effects of TPPs are dose-dependent, and that ST extract is the least cytotoxic TPP tested in this study.     

Investigation on cobalt‐oxide nanoparticles cyto‐genotoxicity and inflammatory response in two types of respiratory cells

The increasing use of cobalt oxide (Co₃O₄) nanoparticles (NPs) in several applications and the suggested genotoxic potential of Co‐oxide highlight the importance of evaluating Co₃O₄ NPs toxicity. Cyto‐genotoxic and inflammatory effects induced by Co₃O₄ NPs were investigated in human alveolar (A549), and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1. The physicochemical properties of tested NPs were analysed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cytotoxicity was studied to analyze cell viability (WST1 test) and membrane damage (LDH assay), direct/oxidative DNA damage was assessed by the Formamido‐pyrimidine glycosylase (Fpg)‐modified comet assay and inflammation by interleukin (IL)‐6, IL‐8 and tumor necrosis factor‐alpha (TNF‐α) release (ELISA). In A549 cells, no cytotoxicity was found, whereas BEAS‐2B cells showed a viability reduction at 40 µg ml^–1 and early membrane damage at 1, 5 and 40 µg ml–1. In A549 cells, direct and oxidative DNA damage at 20 and 40 µg ml^–1 were detected without any effects on cytokine release. In BEAS‐2B cells, significant direct DNA damage at 40 µg ml^–1 and significant oxidative DNA damage with a peak at 5 µg ml^–1, that was associated with increased TNF‐α release at 1 µg ml^–1 after 2 h and increased IL‐8 release at 20 µg ml^–1 after 24 h, were detected. The findings show in the transformed alveolar cells no cytotoxicity and genotoxic/oxidative effects at 20 and 40 µg ml^–1. In normal bronchial cells, moderate cytotoxicity, direct DNA damage only at the highest concentration and significant oxidative‐inflammatory effects at lower concentrations were detected. The findings confirm the genotoxic‐oxidative potential of Co₃O₄ NPs and show greater sensitivity of BEAS‐2B cells to cytotoxic and oxidative‐inflammatory effects suggesting the use of different cell lines and multiple end‐points to elucidate Co₃O₄ NPs toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation

A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.     

Cytotoxicity of dental composite (co)monomers and the amalgam component Hg2+ in human gingival fibroblasts

Unpolymerized resin (co)monomers or mercury (Hg) can be released from restorative dental materials (e.g. composites and amalgam). They can diffuse into the tooth pulp or the gingiva. They can also reach the gingiva and organs by the circulating blood after the uptake from swallowed saliva. The cytotoxicity of dental composite components hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and bisglycidylmethacrylate (Bis-GMA) as well as the amalgam component Hg²⁺ (as HgCl₂) and methyl mercury chloride (MeHgCl) was investigated on human gingival fibroblasts (HGFs) at two time intervals. To test the cytotoxicity of substances, the bromodeoxyuridine (BrdU) assay and the lactate dehydrogenase (LDH) assay were used. The test substances were added in various concentrations and cells were incubated for 24 or 48 h. The EC₅₀ values were obtained as half-maximum-effect concentrations from fitted curves. Following EC₅₀ values were found [BrdU: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 8.860 (0.440)/6.600(0.630), TEGDMA 1.810(0.130)/1.220(0.130), UDMA 0.120(0.010)/0.140(0.010), BisGMA 0.060(0.004)/0.040(0.002), HgCl₂ 0.015(0.001)/0.050(0.006), and MeHgCl 0.004(0.001)/0.005(0.001). Following EC₅₀ values were found [LDH: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 9.490(0.300)/7.890(1.230), TEGDMA 2.300(0.470)/1.950(0.310), UDMA 0.200(0.007)/0.100(0.007), BisGMA 0.070(0.005)/0.100(0.002), and MeHgCl 0.014(0.006)/0.010(0.003). In both assays, the following range of increased toxicity was found for composite components (24 and 48 h): HEMA < TEGDMA < UDMA < BisGMA. In both assays, MeHgCl was the most toxic substance. In the BrdU assay, Hg²⁺ was about fourfold less toxic than MeHgCl but Hg²⁺ was about fourfold more toxic than BisGMA. In the BrdU test, a significantly (P<0.05) decreased toxicity was observed for Hg²⁺ at 48 h, compared to the 24 h Hg²⁺-exposure. A time depending decreased toxicity was observed only for Hg²⁺ which can then reach the toxic level of BisGMA.