Article Cite This: Chem. Res. Toxicol. 2018, 31, 594−600
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Assessment of Genotoxic Effects by Constructing a 3D Cellular System with Highly Sensitive Mutagenic Human−Hamster Hybrid Cells Yajun Zhang,†,‡,∥ Shengmin Xu,*,†,∥ Tao Wu,†,‡,∥ Kunyu Hu,⊥ Shaopeng Chen,†,∥ An Xu,†,∥ and Lijun Wu*,†,‡,§,∥
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Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China ‡ University of Science and Technology of China, Hefei, Anhui 230026, China § Institute of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, China ∥ Key Laboratory of Environmental Toxicology and Pollution Control Technology of Anhui Province, Hefei, Anhui 230031, China ⊥ School of Environment Science and Spatial Informatics, China University of Mining and Technology, Xuzhou 221116, China S Supporting Information *
ABSTRACT: Owing to complex microenvironmental conditions, it is challenging to reflect the actual biological responses of tissues or the body in a two-dimensional (2D) cellular system. In the present study, a lowattachment-cultivation technique was employed to establish a highly sensitive 3D human−hamster hybrid (AL) model to study the mutagenic effects of environmental pollutants. The results showed that the established 3D system has apparent organizational characteristics. The average diameter and average cell number of the 3D cells were approximately 240 μm and 1500, respectively. The expression of stemness and cell-junction genes (biomarkers for 3D cells) was higher than that in 2D cells. The present study analyzed the mutagenic effects of the environmental carcinogens arsenite and silver nanoparticles using the established 3D system to demonstrate its efficiency in mutagenic assessment. The results showed that the mutagenic effects of arsenite (10 μM) and silver nanoparticles (10 μg/mL) were 70 ± 3 and 99 ± 7 per 105 survivors, respectively. These values were much lower than those from 2D AL cells and comparable to those from the in vivo system. These results suggest that the developed 3D-cell-culture model based on the 2D AL cellular system more effectively reflects the actual gene-mutation frequency of mutagens in vivo.
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INTRODUCTION Gene mutations induced by chemical carcinogens, radiation, viruses, and others play an important role in the carcinogenic process.1,2 Thus, the development of gene-loci-mutation assays provides reliable data for the cancer-risk assessment of environmental pollutants.3,4 Currently, there are several geneloci-mutation assays to assess the effects of a carcinogenic substance.2,4 Among these methods, the use of human− Chinese-hamster-ovary hybrid (AL) cells, which have a standard set of Chinese-hamster-ovary (CHO) chromosomes with an additional human-fibroblast single chromosome 11, is considered as one of the most sensitive environmentalmutagen-detection models and is widely used in detecting environmental mutagens such as persistent organic pollutants and heavy metals. For instance, Hei et al. reported that mutation-fraction induction with graded doses of sodium arsenite was more sensitive at the CD59 locus than at the HPRT locus.5 Using this mutation-detection system, we also showed that ultraviolet A (UVA) could activate diesel© 2018 American Chemical Society
particulate-extract (DPE)-induced mutagenicity in mammalian cells, and the physicochemical transformation of ZnO nanoparticles (NPs) played a considerable role in ZnONPinduced mutagenicity.6,7 Meanwhile, a major criticism of the 2D cellular system is also proposed, indicating that 2D cultured cells are inadequate representations of living tissue, which often makes them unreliable predictors of in vivo toxicity detection.8 Virtually, all cells in vivo reside in a 3D environment, interacting with the neighboring cells, extracellular matrix (ECM), and growth factors, which is of crucial importance for their proliferation and metabolism. For instance, Lee et al. showed that the biological, histological, and molecular features of traditional 2D techniques were dramatically different from those of primary tissues.9 Breslin and O’Driscoll have elucidated that the cell− cell and cell−extracellular signaling that occur in vivo are Received: March 14, 2018 Published: June 8, 2018 594
DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
Article
Chemical Research in Toxicology essential to cell differentiation, proliferation, and a range of other cellular functions.10 Therefore, there might be a considerable discrepancy between in vitro 2D cell cultures and animal models in terms of many complex biological responses, such as tissue-specific morphology, cell−ECM interactions, cell−cell interactions, and biochemical signals.10,11 To address this problem, an in vitro 3D-cell-culture technique, an artificially created environment for biological cells to grow or interact with their surroundings in all three dimensions that mimics tissue integrity has been proposed and developed for simulating the cellular characteristics of a microenvironment in vivo.12 It bridges the gap between in vivo animal models and in vitro 2D cell cultures. Researchers have shown that 3D cells closely reflect the phenotype and genotype of primary tissues.9,13,14 For example, Lee et al. showed that 3D cells could reflect the biological, histological, and molecular features of primary tumors.9 To more reliably embody the mutagenic response of environmental pollutants, we developed a 3D-cell-culture model based on 2D AL cells using a lowattachment-cultivation technique and assessed the genemutation effects of sodium arsenite (NaAsO2) and silver nanoparticles (AgNPs). Our results found that the established 3D model was more significant and reliable than the traditional monolayer AL culture for mutagenic-risk assessment of environmental pollutants.
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cell count (cells per milliliter) number of live cells counted = × 10 000 number of large corner squares counted
(1)
To count the number of cells, 3D spheres were transformed into single-cell suspensions, and the numbers were calculated using the equation below: cell count (cells per sphere) total number of cells counted = × 10 000 number of squares
(2)
Gene-Expression Analysis in 3D Cell Cultures. The geneexpression levels were measured using quantitative RT-PCR. Total RNAs of monolayer and sphere cells were isolated using Trizol reagent (Invitrogen). Next, 3 mg of total RNA was added to reverse transcription kits (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, TransGen). A StepOnePlus Real-Time PCR System (Applied Biosystems) was utilized to conduct qRT-PCR. The expression level was analyzed by using the ΔΔCT method and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for each cDNA sample. Primer sequences were listed in Table S1. Cell-Colony Formation and CD59-Gene-Loci-Mutation Assay. Overall cell survival was assessed by cell-colony formation by plating 300 cells in 60 mm cell-culture dishes in triplicate. After 8 days, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet, and the surviving colonies were counted. The survival rate was calculated as the total number of the treated group divided by the total number of the control group. The number of colonies was counted to determine the surviving fraction as described.7 Mutagenicity testing of AL cells was carried out as previously described.7 After the various treatments, an expression period of 1 week was necessary for the cells to recover from the temporary growth lag. To determine the mutant rate, 2 mL of complete Ham’s F12 medium containing 5 × 104 cells were plated into 60 mm dishes. After incubation for 4 h, 0.2% CD59 antiserum and 1.5% (v/v) fresh complement were added to each dish; 300 cells containing complement alone with no additional agent were used as the control. The cultures were further incubated for another 8 days. After that, the cells were fixed, stained, and counted. Mutagenic Effects of AgNPs and NaAsO2 in a 3D AL Cellular System. Cells from both monolayer cultures and spheres were seeded in Petri dishes or low-attachment dishes and incubated for 24 h. Then, the indicated concentrations of AgNPs or NaAsO2 were added. After 24 h, the AgNP- and NaAsO2-treated groups and the control group were subject to the mutagenicity-testing assay. Quantitative Measurement of Cellular Uptake with AgNPs and NaAsO2. AL cells treated with 50 μg/mL AgNPs or 50 μM NaAsO2 for 2 h were collected and incubated in HNO3 for 24 h at room temperature. Ag content inside cells was measured using inductively coupled plasma mass spectrometry (ICP-MS; Thermo XSeries II, Thermo Scientific) and arsenic content inside cells was measured by atomic-fluorescence spectrometry (AFS; AFS-9130, Titan Instruments). Data Analysis. All data are from three to four independent experiments and are expressed as the means ± SD. One-way analysis of variance (ANOVA) was used to analyze the mean differences of the treatments groups compared with the control groups, and two-way ANOVA was used to compare the mean differences among the groups. The criterion for statistical significance was p < 0.05.
MATERIALS AND METHODS
Monolayer Cell Culture. AL cells were cultured in Ham’s F12 medium (Hyclone) supplemented with 8% fetal bovine serum (FBS, Biowest), 2 × 10−4 M glycine, and 25 μg/mL gentamicin as previously described.15 The culture medium was changed every 2 days. In all experiments, cells were maintained in a humidified 37 °C incubator with 5% CO2. Construction of 3D Spheres. The low-attachment-cultivation technique for 3D cell cultures was carried out as described previously.16,17 Briefly, equal volumes of 1% agarose solution and 2 × DMEM medium were mixed in a cell-culture dish (Corning). The mixture became gelatinoid after the temperature decreased to room temperature. Agarose solution (1%) was made with agarose (Biowest) and sterilized by heating to 121 °C for 30 min. DMEM medium (2×) was prepared by dissolving 7.4 g of sodium bicarbonate and 26.8 g of DMEM powder (high glucose, Gibco) in 1000 mL of deionized water and sterilized with a 0.22 μm filter. Cell spheres were cultured in lowattachment dish at a density of 3 × 106 cells/dish. The culture medium was changed every 2 days. Conversion of 3D Spheres to Monolayers. Cells were recovered from 3D spheres using trypsin−EDTA solution according to a previously described method.18 In brief, 3D cultures were first submerged in medium for 20 to 30 s in a centrifuge tube; then, 3D cultures were sequentially sieved through a cell strainer with 60 and 180 mesh numbers to achieve suitable cell masses and resuspended in PBS. The cell suspension was then centrifuged at 800 rpm for 3 min, and the supernatant was discarded. To make single-cell suspensions, 3D spheres were trypsinized using trypsin−EDTA and centrifuged. The supernatant was discarded, and the cells were replated in complete Ham’s F12 medium. Analysis of the Size of the 3D Spheres. Morphology of 3D spheres, which had the photomicrographic appearance of almost perfect spheres, was projected by means of an Olympus IX71 microscope (Olympus). The diameters of the 3D spheres in the photomicrographs could be measured by Cellsens Standard software (Olympus). Monolayer adherent cells were brought into suspension using trypsin−EDTA and resuspended in a volume of fresh Ham’s F12 medium. The cell numbers were counted by hemocytometer, and the concentration of cells was calculated using the equation below:
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RESULTS Dynamic Observation and Characterization of the 3D-Sphere System. Low-attachment cultivation is a technique in which cells can be grown on a favorable scaffold to form a sphere. On the basis of this technique, 3D spheres were developed using AL cells. Cell morphology was observed to analyze the dynamics of sphere formation. At earlier points, 595
DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
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Chemical Research in Toxicology
Figure 1. Dynamic observation of sphere formation. (A) Morphology of the cultured AL cells captured under a differential-interference-contrast (DIC) microscope. (B) 3D cell diameters and numbers per sphere calculated from the consecutive culture.
Figure 2. Morphological features and biomarker expressions of organotypic tissue in 3D cultured cells. (A) qRT-PCR analysis of Oct4 expression on consecutive days of culture. (B) qRT-PCR analysis of Nanog, Oct4, Sox2, and Lin28 in AL cells in monolayer and sphere culture. (C) qRT-PCR analysis of Cx43 and LI-cadherin in monolayer and sphere culture. **p < 0.01, ***p < 0.001.
cells tended to exhibit loose cellular aggregates in which individual cells could be distinguished clearly (Figure S1). The morphology of the 3D sphere formed after 8 days of incubation maintained stable 3D morphology even several days later. 3D cultures were sequentially sieved through a cell strainer to achieve uniform 3D spheres (Figure 1A. The morphology of these cultures was further characterized by the sphere diameter and the number of cells per 3D structure. After the cells were cultured for 8 consecutive days, the average diameter of the 3D spheres was found to be approximately 240 μm using the Cellsens Standard software (Olympus), and the average cell number of the 3D spheres was found to be approximately 1500 (Figure 1B). The diameters and cell numbers of 3D spheres did not further increase significantly with increasing incubation time. Biochemical Characterization between 2D and 3D Cultured Cells. Previous studies indicated that cell aggregates
tend to help maintain stemness. To verify this, we detected the expression of several stemness-marker genes in 3D spheres. The expression of stemness-marker genes continually increased with time, reaching the maximum value on the eighth day of incubation, and was then followed by a gradual slowdown, which represented the formation of a large, dense organization; the expression of Oct4 is shown as an example in Figure 2A. Moreover, at 8 days, Oct4, Nanog, Sox2, and Lin28 expression was significantly elevated in 3D spheres compared with in 2D monolayer cultures (Figure 2B). Additionally, we also analyzed the expression of Connexin 43 (Cx43) and LI-cadherin. Cx43 is a member of structurally related transmembrane proteins, and LI-cadherin is a classic cadherin that mediates intercellular adhesion. Both of them act as scaffolds for the organization of transmembrane-junction proteins and enlist a variety of signaling molecules for specific functions.19 The study showed that closer interactions among cells in the 3D cultures might 596
DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
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Chemical Research in Toxicology
Figure 3. Mutagenic background of the 3D AL system at the CD59 locus. (A) Surviving fraction of 3D AL cells on consecutive days of culture. (B) Mutation background of AL cells at the CD59 gene locus on consecutive days of culture.
Figure 4. Evaluation of NaAsO2 and AgNP toxicity in 3D spheres of AL cells and 2D AL cells. (A,C) Effects of NaAsO2 (A) and AgNPs (C) on surviving fractions. (B,D) Mutagenicity induced by NaAsO2 (B) and AgNPs (D). (E) Quantitative measurement of cellular uptake of AgNPs and NaAsO2. (F) HIS image of the distribution of AgNPs in 2D and 3D cells. *p < 0.05, **p < 0.01, ***p < 0.001.
trigger higher expression of cell junction genes, including Cx43 and LI-cadherin.20 As shown in Figure 2C, high expression levels of Cx43 and LI-cadherin were detected in our 3D model. Collectively, these results indicate that 3D AL spheres cultivated using the low-attachment-cultivation method formed tissue-specific structures in 8 days. Viability and Mutagenic Background of the 3D AL System at the CD59 Locus. The key element of a sensitive mutagenic system is to keep the CD59 spontaneous mutation at low levels. For the 2D AL system, the plating efficiency and mutation background are approximately 80.67 ± 5.02% and 50 ± 7 mutants per 105 survivors, respectively. After establishment of the 3D AL models, we detected the survival fraction and mutation background of the 3D spheres on different culture days. With a cell-colony-formation assay, we demonstrated that the cellular survival of the 3D system remains unchanged during culture periods (Figure 3A). Similarly, mutation induction at the CD59 locus of the 3D spheres was similar to that of 2D cells during the culture period of 4 to 8 days
(Figure 3B). On the basis of these results and the expression of marker genes, 3D spheres cultured for 8 days were subsequently chosen for further experiments. Mutagenicity Assessment of NaAsO2 and AgNPs in 3D Spheres. With the established 3D-AL-cell-culture model, we studied the mutagenic effects of NaAsO2, a well-known human carcinogen, and AgNPs, a widely used nanomaterial. As shown in Figure 4, both NaAsO2 and AgNPs could significantly increase the mutation fraction of 2D cells but not that of 3D spheres (Figure 4). Compared with the untreated group, the surviving fraction of 2D AL cells treated with 1 μM NaAsO2 was 87.34 ± 3.59%, and the value decreased to 59.38 ± 1.64% after treatment with 10 μM NaAsO2. The surviving fractions of 2D AL cells after NaAsO2 treatment were found to be significantly lower than those of 3D cells, which were 89.23 ± 5.20% with 1 μM NaAsO2 treatment and 77.24 ± 3.38% with 10 μM NaAsO2 treatment (Figure 4A). Meanwhile, NaAsO2 (10 μM) could induce CD59 mutation in both 2D and 3D cells with mutation 597
DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
Article
Chemical Research in Toxicology fractions of 134 ± 5 and 70 ± 3 per 105 survivors, respectively (Figure 4B). The same phenomenon was again observed in the treatment with AgNPs. At AgNP concentrations of 1 and 10 μg/mL, the survival fractions of 2D AL cells decreased to 89.59 ± 4.81 and 14.69 ± 3.67%, respectively, and the survival fractions of 3D cells decreased to 84.94 ± 4.42 and 59.60 ± 5.01%, respectively. After AgNP treatment, the 2D AL cells had significantly lower survival fractions than those of the 3D cells. Mutation fractions of 2D and 3D cells upon addition of 10 μg/ mL AgNPs were found to be 169 ± 12 and 99 ± 7 per 105 survivors, respectively (Figure 4C,D). To identify the possible reason for this phenomenon, we used AFS to detect arsenic uptake by 2D and 3D cells. It was found that the amount of arsenic in 2D cells was significantly higher than that in 3D cells (Figure 4E). To quantify Ag ions, we used ICP-MS. We found that the amount of Ag was much higher in 2D cells than in 3D cells (Figure 4E). A hyperspectral-imaging system (Olympus, BX51, HIS) was further used to image Ag distribution in 2D and 3D cells. Figure 4F shows that AgNPs could be easily identified from their spectral signature in 2D cells exposed to 10 μg/mL AgNPs for 24 h. However, this metallic luster was less obvious in 3D cells compared with in 2D cells (Figure 4F). These results provided evidence that the 3D multilayered spheres had reduced uptake of environmental pollutants to inner cells, which significantly influenced their toxicity. Moreover, the intercellular signaling was different between the 2D and 3D AL cells, which may play another critical role in resistance to toxicity.21
identified aggregation as 3D spheroidal cells on the basis of the characterization of 3D spheres by analysis of morphology, stemness properties, and cell junctions. It was found that only a culture period of 4 days or more could significantly induce LDA formations, which are typically large and tightly packed spheroids. Previous studies demonstrated that the expression of stemness-marker genes is widely used to identify 3D spheres.13,14,16 For instance, Su et al. used the expression of stemness-marker genes as a biomarker to analyze sphere formation.16 Xue et al. claimed that the 3D microenvironment could induce the cells to express these reprogramming transcription factors.25 By RT-qPCR, we found that these reprogramming factors were significantly upregulated in 3D cell cultures compared with in monolayer 2D cultures. Furthermore, we also analyzed the expression of genes encoding cellular-adhesion molecules that act as scaffolds, promoting closer contact between cells and autoregulating a variety of signaling pathways.17 We found that 3D AL cells expressed LI-cadherin and Cx43. Similar to our study, Asaithamby et al. also characterized the 3D structure of human bronchial epithelial cells in Matrigel for 6 days by immunostaining with antibodies specific to E-cadherin.18 These results demonstrate that AL cells develop into 3D spheres in low-attachment-cultivation dishes after a culture period of 8 days. After 3D spheres were obtained, we used a cell strainer to achieve uniform 3D spheres to improve the reproducibility of the experiment. Multicellular spheroids often have a core of dead cells surrounded by an outer layer of living cells that display significant gradients of critical metabolites such as oxygen, glucose, other nutrients, and growth factors. Hypoxia occurs in microregions of larger tumors that are 150−200 μm away from the exterior cells.26 Our results also indicate that compared with monolayer cells, the cellular viability and mutagenic background of extremely large aggregates (ELAs) are significantly different (Figure S2). Therefore, LDAs cultured for 8 days were chosen for subsequent mutagenic experiments. We compared the mutagenicity of NaAsO2 and AgNPs in both 2D AL cells and 3D spheres. Arsenite has been reported to be a mutagen based on reactive-oxygen-species generation.5 Kumar demonstrated that arsenic trioxide significantly induced DNA damage in a dose-dependent manner in HL-60 cells, with the lowest concentration at 2 μg/mL.27 Consistent with these findings, NaAsO2 was found to significantly increase the mutation induction of 2D cells. However, the genotoxicity of arsenic in 3D spheres was much lower than that in 2D cells. Similar results were found in the case of treatment with AgNPs (Figure 4). In the 3D system, no apparent toxicity effects were observed, whereas AgNPs could significantly increase the genotoxicity of 2D cells. The enhanced toxicity resistance in 3D models might be attributed to the multilayered structure of the spheroid culture, which can reduce penetration of toxicants into the central areas. Moreover, there might also be diffusion of NPs into the spheroid upon AgNP treatment. To validate this hypothesis we detected the penetration of AgNPs into 3D spheres using HIS, which can track and identify specific materials on the basis of their light-scattering spectra. As shown in Figure 4F, AgNPs are harder to distinguish in 3D spheres, but this metallic luster was more obvious in 2D cells. Additionally, we used ICP-MS to detect Ag ions and AFS to detect arsenic. Both Ag ions and arsenic were much higher in 2D cells than in 3D cells, demonstrating that the multilayered structure leads to the
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DISCUSSION Since the development of a method to cultivate live cells in vitro by Harisson in 1907, massive advances have been made in cell-culture techniques. Thousands of published studies in biology have relied on these techniques.22,23 Meanwhile, a major criticism of these studies is that there is a large discrepancy between in vitro 2D cell cultures and animal models, concerning many complex biological responses such as tissue-specific morphology, cell−ECM interactions, cell−cell interactions, and biochemical signals.10,11 Researchers also state that the issue of toxicity-test results varying quite significantly among different culture models has now become apparent. Genotoxicity assays of environmental pollutants using 2D-cell-culture models show highly adverse effects, but no apparent toxicity effects are observed in animal models.24 For these reasons, in vitro 3D cell culture has been proposed and developed for simulating the cellular characteristics of a microenvironment in vivo. However, there is a research gap in the 3D cellular system for genotoxicity screening, which is crucial for bridging in vivo animal models on one end and in vitro monolayer cell culture on the other. In the present study, we developed a 3D-cell-culture model based on the 2D AL mutation-detection system to reflect the genotoxic effect of mutagens in vivo effectively. To identify a 3D-cell-culture model, three parameters need to be tested: (1) morphology, (2) expression of key transcription factors for cell reprogramming and, (3) expression of cell junctions. Lee et al. employed phase-contrast microscopy to analyze live-cell structures and classified 3D spheroid morphologies into three distinct stages: small aggregates (SAs), large loose aggregates (LLAs), and large dense aggregates (LDAs).9 Using soft agarose as a lowattachment surface for engineering our 3D AL model, we 598
DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
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Chemical Research in Toxicology
nexin 43; SAs,small aggregates; LLAs,large loose aggregates; LDAs,large dense aggregates; ELAs,extremely large aggregates
observed toxicity resistance to AgNPs in 3D cultured cells. The data obtained from toxicity experiments on 3D cells was more realistic, reflecting the pollutants’ toxicity in vivo. The study also showed that the administration of water containing 85 ppm NaAsO2 (approximately 650 μM, far above the arsenic concentration levels in 2D cells) to pregnant mice during the gestation period of 8 to 18 days did not induce livers tumorigenesis in their offspring when examined at 6 weeks or 49 weeks of age.28 Also, studies on activity testing of nanomaterials, environmental pollutants, and drugs using 2Dcell-culture models showed highly adverse effects, but no apparent toxicity effects were observed in animal models.24,29,30 In summary, 3D spheres of AL cells were obtained by utilizing a low-attachment-cultivation technique. The results demonstrated that agarose might facilitate tumorigenesis by providing a semblable tumor environment. These cells were reprogrammed and acquired complex biological responses to various degrees via this physical method without using any exogenous additives. 3D-sphere-culture models could provide a better understanding of the mutagenic effects of environmental pollutants and radiation in vivo and may serve as a safer and more effective technique to evaluate genotoxicity than other physical approaches.
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(1) Chen, J., Miller, B. F., and Furano, A. V. (2014) Repair of naturally occurring mismatches can induce mutations in flanking DNA. eLife 3, e02001. (2) de Morais, C. R., Bonetti, A. M., Carvalho, S. M., de Rezende, A. A. A., Araujo, G. R., and Spano, M. A. (2016) Assessment of the mutagenic, recombinogenic and carcinogenic potential of fipronil insecticide in somatic cells of Drosophila melanogaster. Chemosphere 165, 342−351. (3) Atienzar, F. A., and Jha, A. N. (2006) The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: A critical review. Mutat. Res., Rev. Mutat. Res. 613, 76−102. (4) Pan, X., Redding, J. E., Wiley, P. A., Wen, L., McConnell, J. S., and Zhang, B. (2010) Mutagenicity evaluation of metal oxide nanoparticles by the bacterial reverse mutation assay. Chemosphere 79, 113−116. (5) Hei, T. K., Liu, S. X., and Waldren, C. (1998) Mutagenicity of arsenic in mammalian cells: Role of reactive oxygen species. Proc. Natl. Acad. Sci. U. S. A. 95, 8103−8107. (6) Bao, L. Z., Xu, A., Tong, L. P., Chen, S. P., Zhu, L. Y., Zhao, Y., Zhao, G. P., Jiang, E. K., Wang, J., and Wu, L. J. (2009) Activated Toxicity of Diesel Particulate Extract by Ultraviolet A Radiation in Mammalian Cells: Role of Singlet Oxygen. Environ. Health Perspect. 117, 436−441. (7) Wang, M. M., Wang, Y. C., Wang, X. N., Liu, Y., Zhang, H., Zhang, J. W., Huang, Q., Chen, S. P., Hei, T. K., Wu, L. J., and Xu, A. (2015) Mutagenicity of ZnO nanoparticles in mammalian cells: Role of physicochemical transformations under the aging process. Nanotoxicology 9, 972−982. (8) Sayes, C. M., Reed, K. L., and Warheit, D. B. (2007) Assessing toxicity of fine and nanoparticles: Comparing in vitro measurements to in vivo pulmonary toxicity profiles. Toxicol. Sci. 97, 163−180. (9) Lee, J. M., Mhawech-Fauceglia, P., Lee, N., Parsanian, L. C., Lin, Y. G., Gayther, S. A., and Lawrenson, K. (2013) A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro. Lab. Invest. 93, 528−542. (10) Breslin, S., and O’Driscoll, L. (2013) Three-dimensional cell culture: the missing link in drug discovery. Drug Discovery Today 18, 240−249. (11) Haycock, J. W. (2011) 3D Cell Culture: A Review of Current Approaches and Techniques. Methods Mol. Biol. 695, 1−15. (12) Huh, D., Hamilton, G. A., and Ingber, D. E. (2011) From 3D cell culture to organs-on-chips. Trends Cell Biol. 21, 745−754. (13) Ghosh, S., Spagnoli, G. C., Martin, I., Ploegert, S., Demougin, P., Heberer, M., and Reschner, A. (2005) Three-dimensional culture of melanoma cells profoundly affects gene expression profile: A high density oligonucleotide array study. J. Cell. Physiol. 204, 522−531. (14) Kenny, P. A., Lee, G. Y., Myers, C. A., Neve, R. M., Semeiks, J. R., Spellman, P. T., Lorenz, K., Lee, E. H., Barcellos-Hoff, M. H., Petersen, O. W., Gray, J. W., and Bissell, M. J. (2007) The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol. Oncol. 1, 84−96. (15) Zhao, G. P., Wang, J., Wang, X. F., Chen, S. P., Zhao, Y., Gu, F., Xu, A., and Wu, L. J. (2011) Mutagenicity of PFOA in Mammalian Cells: Role of Mitochondria-Dependent Reactive Oxygen Species. Environ. Sci. Technol. 45, 1638−1644. (16) Su, G. N., Zhao, Y. N., Wei, J. S., Han, J., Chen, L., Xiao, Z. F., Chen, B., and Dai, J. W. (2013) The effect of forced growth of cells into 3D spheres using low attachment surfaces on the acquisition of sternness properties. Biomaterials 34, 3215−3222. (17) Carvalho, M. R., Lima, D., Reis, R. L., Correlo, V. M., and Oliveira, J. M. (2015) Evaluating Biomaterial- and Microfluidic-Based 3D Tumor Models. Trends Biotechnol. 33, 667−678. (18) Asaithamby, A., Hu, B. R., Delgado, O., Ding, L. H., Story, M. D., Minna, J. D., Shay, J. W., and Chen, D. J. (2011) Irreparable
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00069. Morphology of cultured AL cells captured under DIC microscope; morphology, viability, and mutagenic background of ELAs; and sequences of PCR primers used in our study (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Tel.: 86-551-65591602. Fax: 86-551-65595670. E-mail:
[email protected] (S.M.X.). *E-mail:
[email protected] (L.J.W.). ORCID
Shengmin Xu: 0000-0002-4118-3419 Lijun Wu: 0000-0002-5005-6403 Funding
This study was supported by the Strategic Priority Research P r o g r a m of t h e C h i n e s e A c a d e m y o f S c i e n c e s (XDB14030502), the National Basic Research 973 Program (2014CB932002), and the National Natural Science Foundation of China (31470829 and 81273004). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS AL,human−Chinese-hamster-ovary hybrid; CHO,Chinese hamster ovary; UVA,ultraviolet A; DPE,diesel-particulate extract; NPs,nanoparticles; ECM,extracellular matrix; NaAsO2,sodium arsenite; AgNPs,silver nanoparticles; FBS,fetal bovine serum; GAPDH,glyceraldehyde 3-phosphate dehydrogenase; ICP-MS,inductively coupled plasma mass spectrometry; AFS,atomic-fluorescence spectrometry; ANOVA,analysis of variance; DIC,differential interference contrast; Cx43,Con599
DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
Article
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DOI: 10.1021/acs.chemrestox.8b00069 Chem. Res. Toxicol. 2018, 31, 594−600
