Genotoxicity Assessment of Mesoporous Silica and Graphene Oxide in GDL1 Cells

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However, their potential genotoxicity remains poorly understood. To evaluate the associated health risks of mesoporous silica and graphene oxide, we assessed their cytotoxicity and genotoxicity in GDL1 cells using trypan blue exclusion and gpt mutation assays, followed by mutation frequency and spectrum analysis through gpt gene sequencing. Results A 24-hour exposure of mesoporous silica to GDL1 cells induced dose-dependent reductions in cell viability, as well as dose-dependent increases in gpt mutation frequencies at 0.06 and 0.09 mg/mL. Graphene oxide induced cytotoxicity at higher concentrations (0.2 and 0.4 mg/mL) and significantly increased gpt mutation frequency in the highest concentration exposure group compared to controls. Mutation spectrum analysis revealed a significant increase in G:C to A:T transitions in both the exposed groups. In addition, exposure to mesoporous silica significantly increased G:C to T:A transversions, while graphene oxide exposure significantly increased G:C to C:G transversions. Mutation hotspots at positions 64, 164, and 416 were identified exclusively in the mesoporous silica-treated group, indicating material-specific mutagenesis. Mutation hotspot at position 401 was detected exclusively in the graphene oxide group, indicating this site as a potential mutation hotspot. Conclusion These results demonstrate that both mesoporous silica and graphene oxide exhibit cytotoxic and genotoxic potential in vitro . The mutation patterns suggest that oxidative DNA damage and inflammation may contribute to the observed genotoxicity. Further investigations are necessary to elucidate the molecular mechanisms underlying the mutagenicity of these nanomaterials and their implications for human health risk assessment. Mesoporous silica Graphene oxide Organoid Genotoxicity Figures Figure 1 Figure 2 Figure 3 Introduction Nanomaterials are defined as materials with at least one dimension in the size range from approximately between 1 and 100 nm and have unique physical, biological, and chemical characteristics [ 1 ]. A wide variety of nanomaterials have already been used for several decades and are applied in various applications for industrial, medical, and cosmetic fields because of their useful properties. Among the various nanomaterials, mesoporous silica has attracted a great deal attention due to its exceptional properties such as uniform pore size, very large surface area, ease of surface modification, high biocompatibility and biodegradability, high mechanical and thermal stability and stable aqueous dispersibility [ 2 , 3 ]. These advantageous properties make mesoporous silica highly suitable for applications in catalysis, drug delivery systems (DDS), and diagnostic imaging [ 4 , 5 ]. Graphene oxide has also garnered considerable interest in biological and medical research due to its unique physical and chemical properties [ 6 ]. Graphene oxide is a material that can bond with oxygen-containing functional groups (epoxy, hydroxy, carboxyl, and carbonyl groups) on its surface and has attracted considerable attention [ 7 ]. These functional groups facilitate surface modifications, allowing for the attachment of various biomolecules (e.g., proteins, DNA, RNA) and chemical compounds. Consequently, graphene oxide has been utilized as a biosensor surface and in DDS platforms [ 8 , 9 , 10 , 11 ]. Other biomedical applications include photothermal therapy [ 12 ] and tissue engineering [ 13 ]. Given the increasing application of these substances for human use, their potential release into the environment, and subsequent human exposure, the evaluation of their human health effects has become a key focus in recent years. However, with limited knowledge regarding their safety, there is an urgent need for a human health risk assessment of these materials. Previously, we have examined the genotoxicity of some nanomaterials by using both in vitro and in vivo assays and reported genotoxic and other toxic properties of them [ 14 , 15 , 16 , 17 ]. In the field of genetics, genotoxicity is an important word and refers to the property of chemical agents that are able to alter genetic information. One of the major endpoints of genotoxicity is gene mutation. When a mutation is present in a germ cell, it can be inherited and potentially lead to genetic disorders in the next generation. In somatic cells, mutations in critical genes may lead to cancer development. Thus, every mutagen is considered potentially carcinogenic. Despite the increasing application of mesoporous silica and graphene oxide, their genotoxic potential remains poorly understood. Therefore, it is necessary to determine whether these nanomaterials can induce mutations, and to elucidate the mechanisms underlying their genotoxicity. Major genotoxic events induced by chemicals are point mutations and deletion mutations. In particular, deletion mutations constitute an important class of mutations because they are associated with a wide range of human diseases, including cancer. To assess such mutations in vitro , the gpt delta L1 (GDL1) cell line was established by Takeiri et al . from lung fibroblasts of gpt delta transgenic mice through transfection with the SV40 T antigen [ 18 ]. This cell line is a valuable tool for the detection of both point mutations and deletions, similar to gpt delta transgenic mouse models [ 18 ]. In the present study, we evaluated the cytotoxicity and genotoxicity of mesoporous silica and graphene oxide using the GDL1 cell line, aiming to obtain fundamental data for elucidating their potential health risks. Materials and methods Materials Mesoporous silica (Silica, mesostructured, MCM-41; Sigma-Aldrich, St. Louis, MO, USA) was kindly provided by the National Institute of Health Sciences (Kanagawa, Japan). It consists of silicon dioxide and has an ordered mesoporous structure with pore diameters ranging from 2.1 to 2.7 nm and a surface area of approximately 1,000 m²/g. The mesoporous silica was suspended in ultrapure water (NACALAI TESQUE, INC., Kyoto, Japan) at a concentration of 0.5 mg/mL using an ultrasonic homogenizer (TAITEC Corporation., Saitama, Japan) operated at 40 W for 5 minutes and then diluted to the required concentrations with medium. Graphene oxide (Sigma-Aldrich, USA) was also provided by the National Institute of Health Sciences. It was dispersed in water at a concentration of 4 mg/mL and diluted to the required concentrations using medium by pipetting. Cell culture GDL1 cells were established from gpt delta mice lung fibroblasts [ 18 ] and kindly gifted by Dr Akira Takeiri (Chugai Pharmaceutical., Tokyo, Japan). Cells were passaged every 3–4 days, and cells were dissociated into single cells by treatment with trypsin (Innovative Cell Technologies, San Diego, CA) for 5 min at 37°C at each passage. The maintenance medium was DMEM (high glucose) (NACALAI TESQUE, INC., Kyoto, Japan) containing 10% fetal bovine serum (Gibco,. Carlsbad, CA, USA) and antibiotics (100 units/mL penicillin G and 100 mg/mL streptomycin sulfate, Gibco). We also confirmed that the GDL1 cells were able to be recultured after freezing in LaboBanker 2 (Juji Field, Tokyo, Japan) and storage at − 80°C followed by thawing. Evaluation of cell viability of nanomaterials The effects of mesoporous silica and graphene oxide on the viability of GDL1 cells were evaluated using the trypan blue exclusion method. Briefly, cells (1.0×10 6 cells/well) were incubated for 4–6 hours and then exposed to various concentrations of nanomaterials for 24 hours at 37°C. Cells dissociated with trypsin were treated with 0.4 (w/v) trypan blue solution (Sigma-Aldrich, USA) at a 1:1 ratio and counted using a hemocytometer under a microscope (Olympus Corporation, Tokyo, Japan). gpt mutation assay For mutation analysis, GDL1 cells were exposed to appropriate concentrations of nanomaterials for 24 h. After exposure, nanomaterials were removed, and the cells were washed with PBS continuously, trypsinized and reseeded for subculture. Cells were subsequently harvested and stored at − 80°C until DNA isolation for mutation assay. High-molecular-weight genomic DNA was extracted from GDL1 cells using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instructions. Lambda EG10 phages were rescued using the Transpack Packaging Extract (Stratagene, La Jolla, CA, USA). The gpt mutation assay was performed according to previously described methods (Fukai et al., 2018). Briefly, E. coli YG6020 was infected with the phage and spread on M9 salt plates containing chloramphenicol (Cm) and 6-thioguanine (6-TG). Plates were incubated for 72 h at 37°C to select colonies with a plasmid carrying the gene encoding chloramphenicol acetyltransferase, as well as mutated gpt . The 6-TG–resistant isolates were cultured overnight at 37°C in LB broth containing 25 mg/mL of Cm, harvested by centrifugation (7,000 rpm, 10 min), and stored at − 80°C. Mutational spectra of 6-TG coding sequences were determined using PCR and direct sequencing, and a 739-bp DNA fragment containing gpt was amplified by PCR as described previously [ 19 ]. Sequence analysis was performed using Eurofins Genomics software (Tokyo, Japan) Statistical analysis For the statistical comparison of the experimental and control groups in the gpt mutation assays, data were expressed as means ± standard deviations. The F -test was initially performed to evaluate equality of variances. If variances were unequal, Welch’s t -test was applied. If not, Student’s t- test was used instead. In the case of the mutation spectrum analysis, P -values were determined using Fisher’s exact test according to Carr and Gorelick [ 20 ]. In any case, p -values lower than 0.05 were considered to indicate statistical significance. RESULTS Cytotoxicity of nanomaterials A 24-hour treatment with mesoporous silica resulted in a concentration-dependent decrease in cell viability, with significant growth inhibition observed at concentrations of 0.09, 0.13, and 0.25 mg/mL compared with the vehicle control (Fig. 1a). Exposure to graphene oxide showed no cytotoxicity at 0.1 mg/mL, with cell viability comparable to that of the control (Fig. 1b). However, cell viability decreased to 57% at 0.2 mg/mL exposure group ( p < 0.05 vs control) and 70% in the 0.4 mg/mL exposure group (Fig. 1b). Although a clear concentration-dependent trend was not observed, cytotoxicity was observed in these exposure groups. Based on these results, the concentrations selected for the gpt mutation assay were 0.06 and 0.09 mg/mL for mesoporous silica, and 0.2 and 0.4 mg/mL for graphene oxide. In vitro mutagenicity of nanomaterials The gpt mutation frequencies (MFs) in GDL1 cells exposed to mesoporous silica (0.06 and 0.09 mg/mL) increased compared with the control in a dose-dependent manner (Fig. 2a). The MF in the control group was 19.1 × 10⁻ 6 , whereas it was 16.9 × 10⁻⁶ and 101 × 10⁻⁶ in the 0.2 and 0.4 mg/mL of graphene oxide exposure groups, respectively, with a statistically significant increase observed at the higher concentration (Fig. 2b). The classes of gpt mutations induced by mesoporous silica are summarized in Table 1. Among the mutation profiles observed in the gpt coding sequence in GDL1 cells, the proportion of G:C to A:T transitions significantly increased from 14.3% in the control group to 24.6% in the mesoporous silica exposure group. In addition, exposure induced an increase in G:C to T:A transversions from 0% in the control group to 11.5%. Table 2 summarizes the classes of gpt mutations observed in GDL1 cells exposed to graphene oxide. At 0.4 mg/mL, both G:C to C:G transversions and G:C to A:T transitions were significantly elevated, with the frequency of G:C to C:G transversions increasing from 5.6% in the control group to 20.6% in the exposure group, and the proportion of G:C to A:T transitions increasing from 11.1% to 26.5%. The distribution of nanomaterial-induced and spontaneous mutations in the coding region of gpt is shown in Fig. 3. Of the 53 mutations induced by mesoporous silica, 10 were G to A transitions and 9 were T to G transversions. Mutation hotspots at positions 64, 164, and 416 were exclusively detected in the mesoporous silica-exposed group, while no such mutations were observed in the control group. Therefore, it is suggested that these mutations can be considered as mesoporous silica induced mutations. Transitions of C to T and transversion of G to C were only observed in the graphene oxide-treated group, therefore it is assumed that these mutations were induced by graphene oxide exposure. Notably, mutations at position 401 were detected exclusively in the graphene oxide group, indicating this site as a potential mutation hotspot. Discussion We investigated the cytotoxicity and genotoxicity of mesoporous silica and graphene oxide using GDL1 cells. Cytotoxicity assays revealed a decrease in cell viability following exposure to both nanomaterials, indicating their cytotoxic potential. The cytotoxicity assay for graphene oxide did not show a clear concentration-dependent effect. This may be attributed to differences in the preparation methods of the exposure solutions: mesoporous silica was dispersed by ultrasonication, while graphene oxide was dispersed by pipetting. The lack of uniform dispersion in the high-dose graphene oxide group may have affected the results. In the in vitro mutagenicity test, mesoporous silica significantly increased G:C to T:A and G:C to A:T base substitution mutations. Graphene oxide exposure led to a significant increase in G:C to A:T and G:C to C:G base substitution mutations. One of the known mechanisms through which nanosized particles exert adverse health effects is their ability to generate reactive oxygen species (ROS). Several studies have indicated that the genotoxicity of fine particulate matter, including nanomaterials, is linked to ROS production [ 21 , 22 ]. For instance, exposure to mesoporous silica has been shown to induce ROS generation and oxidative stress in BEAS-2B cells [ 23 ]. ROS are known to cause oxidative modifications of DNA bases. Among the four DNA bases, guanine is particularly susceptible to oxidation, leading to the formation of 8-oxo-7,8-dihydroguanine (8-oxoguanine). During DNA replication, 8-oxoguanine can mispair with adenine, resulting in G:C to T:A transversions [ 24 ]. Therefore, the increase in G:C to T:A transversions caused by mesoporous silica may be attributed to ROS generation. In general, the G:C to C:G transversion is thought to be a rare event in both spontaneous and chemically induced mutations. G:C to C:G transversions are generally considered rare events in both spontaneous and chemically induced mutations. However, Kato et al . reported a significant increase in G:C to C:G transversions in gpt delta transgenic mice exposed to multi-walled carbon nanotubes (MWCNTs), a type of nanocarbon material similar to graphene oxide [ 16 ]. Similar increases in G:C to C:G transversions have also been observed with other nano/microparticles, such as fullerenes (C60), carbon black (CB), and kaolin. In in vitro assay systems, various oxidative stresses caused by sunlight, UV radiation, hydrogen peroxide and peroxyl radicals have frequently been reported to induce G:C to C:G transversions [ 25 , 26 ]. Graphene oxide has also been shown to induce oxidative stress in A549 cells by generating ROS [ 27 ]. While 8-oxo-dG is a common form of DNA damage from oxidative stress, it does not cause G to C transversions because dG is not incorporated opposite 8-oxo-dG [ 28 ]. Instead, other oxidative guanine lesion products—such as imidazolone (Iz), oxazolone (Oz), spiroiminodihydantoin (Sp), and guanidinohydantoin (Gh)—have been identified [ 29 , 30 ]. Among these, Oz, Sp, and Gh are considered key contributors to G to C transversions via translational synthesis mechanisms [ 29 , 30 ]. Therefore, the G:C to C:G transversions induced by graphene oxide may involve the formation of these oxidized guanine derivatives. In this study, G:C to A:T transition mutations were also increased following treatment with both nanoparticles. G to A transitions are commonly observed in both spontaneous and chemically induced mutations and are often associated with deamination of 5-methylcytosine or alkylation of guanine [ 31 , 32 ]. Nitric oxide (NO) causes DNA damage by producing dinitrogen trioxide, which generates diazonium ions that induce DNA deamination and the formation of 8-nitro-dG. This compound can create apurinic sites, preferentially leading to G:C to A:T transition mutations [ 33 , 34 ]. In a previous study, the lungs of rats exposed to mesoporous silica nanoparticles showed a significant increase in NO levels compared to control rats [ 35 ]. Furthermore, graphene oxide has been reported to cause acute lung injury, inflammation, cell apoptosis, and DNA damage through the overproduction of ROS [ 36 ]. Graphene oxide exposure also significantly increased ROS production in mouse embryonic fibroblasts [ 37 ]. These findings suggest that graphene oxide may trigger inflammatory responses and increase NO production. Therefore, both mesoporous silica and graphene oxide may induce G:C to A:T transition mutations via NO generation. Conclusions We demonstrated that mesoporous silica and graphene oxide exhibit cytotoxic and genotoxic potential in an in vitro assay using GDL1 cells, which were established from gpt delta transgenic mice. Based on the prominent mutation spectra observed, it is suggested that oxidative DNA damage and inflammation may be commonly involved in their mutagenicity. However, the detailed mechanisms underlying the genotoxicity of mesoporous silica and graphene oxide were not explored in the present study. Therefore, further investigations are required to elucidate the mechanisms of genotoxicity induced by these materials. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This study was supported by the Japan Agency for Medical Research and Development (AMED), Research on Regulatory Science of Pharmaceuticals and Medical Devices (Grant Number: JP22mk0101247). Author Contribution A.O. and Y.T. designed the study, A.O. acquired funding, R.I. performed the experiments, K.F. and S.H. analyzed the data, and R.I. wrote the initial draft. A.O. and Y.T. reviewed and edited the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. Data availability No datasets were generated or analysed during the current study. References Kohl Y, et al. Genotoxicity of nanomaterials: advanced in vitro models and high-throughput methods for human hazard assessment—a review. Nanomaterials (Basel). 2020;10:1911. Gupta A, Kushwaha SS, Mishra A. A review on recent technologies and patents on silica nanoparticles for cancer treatment and diagnosis. Recent Pat Drug Deliv Formul. 2020;14:126–44. Narayan R, Nayak UY, Raichur AM, Garg S. Mesoporous silica nanoparticles: a comprehensive review on synthesis and recent advances. Pharmaceutics. 2018;10:118. Liu W, Wu J, Jiang Z, Zhang X, Wang Z, Meng F, et al. 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Supplementary Files tables.docx Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Genes and Environment → Version 1 posted Editorial decision: Revision requested 29 Oct, 2025 Reviews received at journal 27 Oct, 2025 Reviews received at journal 24 Oct, 2025 Reviewers agreed at journal 07 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviewers invited by journal 30 Sep, 2025 Editor assigned by journal 30 Sep, 2025 Submission checks completed at journal 30 Sep, 2025 First submitted to journal 22 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":96980,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7684406/v1/eb17361ae384041010e11dac.jpg"},{"id":93529532,"identity":"9b96eb03-e40f-492a-9377-ba3884fd93c9","added_by":"auto","created_at":"2025-10-14 20:51:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66758,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7684406/v1/be984c7adfe9bf6741ab9afa.jpg"},{"id":93530793,"identity":"21fb478e-b17b-4e20-af0c-c47608abdfff","added_by":"auto","created_at":"2025-10-14 21:07:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162566,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7684406/v1/b627a7925224869acb3439ec.jpg"},{"id":99172257,"identity":"4f69c37f-5902-4207-b6cf-c655c4b25d36","added_by":"auto","created_at":"2025-12-29 16:06:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":842029,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7684406/v1/732977bf-c2a1-497b-8811-225bec1786db.pdf"},{"id":93531028,"identity":"5ffbc4e7-27f6-4f52-8f27-2ba3ad2aaed5","added_by":"auto","created_at":"2025-10-14 21:15:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":410715,"visible":true,"origin":"","legend":"","description":"","filename":"tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7684406/v1/aff1fec8ff90374b6787e1ae.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotoxicity Assessment of Mesoporous Silica and Graphene Oxide in GDL1 Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanomaterials are defined as materials with at least one dimension in the size range from approximately between 1 and 100 nm and have unique physical, biological, and chemical characteristics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A wide variety of nanomaterials have already been used for several decades and are applied in various applications for industrial, medical, and cosmetic fields because of their useful properties. Among the various nanomaterials, mesoporous silica has attracted a great deal attention due to its exceptional properties such as uniform pore size, very large surface area, ease of surface modification, high biocompatibility and biodegradability, high mechanical and thermal stability and stable aqueous dispersibility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These advantageous properties make mesoporous silica highly suitable for applications in catalysis, drug delivery systems (DDS), and diagnostic imaging [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Graphene oxide has also garnered considerable interest in biological and medical research due to its unique physical and chemical properties [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Graphene oxide is a material that can bond with oxygen-containing functional groups (epoxy, hydroxy, carboxyl, and carbonyl groups) on its surface and has attracted considerable attention [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These functional groups facilitate surface modifications, allowing for the attachment of various biomolecules (e.g., proteins, DNA, RNA) and chemical compounds. Consequently, graphene oxide has been utilized as a biosensor surface and in DDS platforms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Other biomedical applications include photothermal therapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and tissue engineering [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given the increasing application of these substances for human use, their potential release into the environment, and subsequent human exposure, the evaluation of their human health effects has become a key focus in recent years. However, with limited knowledge regarding their safety, there is an urgent need for a human health risk assessment of these materials.\u003c/p\u003e\u003cp\u003ePreviously, we have examined the genotoxicity of some nanomaterials by using both in \u003cem\u003evitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assays and reported genotoxic and other toxic properties of them [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the field of genetics, genotoxicity is an important word and refers to the property of chemical agents that are able to alter genetic information. One of the major endpoints of genotoxicity is gene mutation. When a mutation is present in a germ cell, it can be inherited and potentially lead to genetic disorders in the next generation. In somatic cells, mutations in critical genes may lead to cancer development. Thus, every mutagen is considered potentially carcinogenic. Despite the increasing application of mesoporous silica and graphene oxide, their genotoxic potential remains poorly understood. Therefore, it is necessary to determine whether these nanomaterials can induce mutations, and to elucidate the mechanisms underlying their genotoxicity.\u003c/p\u003e\u003cp\u003eMajor genotoxic events induced by chemicals are point mutations and deletion mutations. In particular, deletion mutations constitute an important class of mutations because they are associated with a wide range of human diseases, including cancer. To assess such mutations \u003cem\u003ein vitro\u003c/em\u003e, the \u003cem\u003egpt\u003c/em\u003e delta L1 (GDL1) cell line was established by Takeiri \u003cem\u003eet al\u003c/em\u003e. from lung fibroblasts of \u003cem\u003egpt\u003c/em\u003e delta transgenic mice through transfection with the SV40 T antigen [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This cell line is a valuable tool for the detection of both point mutations and deletions, similar to \u003cem\u003egpt\u003c/em\u003e delta transgenic mouse models [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the present study, we evaluated the cytotoxicity and genotoxicity of mesoporous silica and graphene oxide using the GDL1 cell line, aiming to obtain fundamental data for elucidating their potential health risks.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eMesoporous silica (Silica, mesostructured, MCM-41; Sigma-Aldrich, St. Louis, MO, USA) was kindly provided by the National Institute of Health Sciences (Kanagawa, Japan). It consists of silicon dioxide and has an ordered mesoporous structure with pore diameters ranging from 2.1 to 2.7 nm and a surface area of approximately 1,000 m\u0026sup2;/g. The mesoporous silica was suspended in ultrapure water (NACALAI TESQUE, INC., Kyoto, Japan) at a concentration of 0.5 mg/mL using an ultrasonic homogenizer (TAITEC Corporation., Saitama, Japan) operated at 40 W for 5 minutes and then diluted to the required concentrations with medium. Graphene oxide (Sigma-Aldrich, USA) was also provided by the National Institute of Health Sciences. It was dispersed in water at a concentration of 4 mg/mL and diluted to the required concentrations using medium by pipetting.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eGDL1 cells were established from \u003cem\u003egpt\u003c/em\u003e delta mice lung fibroblasts [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and kindly gifted by Dr Akira Takeiri (Chugai Pharmaceutical., Tokyo, Japan). Cells were passaged every 3\u0026ndash;4 days, and cells were dissociated into single cells by treatment with trypsin (Innovative Cell Technologies, San Diego, CA) for 5 min at 37\u0026deg;C at each passage. The maintenance medium was DMEM (high glucose) (NACALAI TESQUE, INC., Kyoto, Japan) containing 10% fetal bovine serum (Gibco,. Carlsbad, CA, USA) and antibiotics (100 units/mL penicillin G and 100 mg/mL streptomycin sulfate, Gibco). We also confirmed that the GDL1 cells were able to be recultured after freezing in LaboBanker 2 (Juji Field, Tokyo, Japan) and storage at \u0026minus;\u0026thinsp;80\u0026deg;C followed by thawing.\u003c/p\u003e\n\u003ch3\u003eEvaluation of cell viability of nanomaterials\u003c/h3\u003e\n\u003cp\u003eThe effects of mesoporous silica and graphene oxide on the viability of GDL1 cells were evaluated using the trypan blue exclusion method. Briefly, cells (1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well) were incubated for 4\u0026ndash;6 hours and then exposed to various concentrations of nanomaterials for 24 hours at 37\u0026deg;C. Cells dissociated with trypsin were treated with 0.4 (w/v) trypan blue solution (Sigma-Aldrich, USA) at a 1:1 ratio and counted using a hemocytometer under a microscope (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003egpt\u003c/b\u003e \u003cb\u003emutation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor mutation analysis, GDL1 cells were exposed to appropriate concentrations of nanomaterials for 24 h. After exposure, nanomaterials were removed, and the cells were washed with PBS continuously, trypsinized and reseeded for subculture. Cells were subsequently harvested and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until DNA isolation for mutation assay. High-molecular-weight genomic DNA was extracted from GDL1 cells using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer\u0026rsquo;s instructions. Lambda EG10 phages were rescued using the Transpack Packaging Extract (Stratagene, La Jolla, CA, USA). The \u003cem\u003egpt\u003c/em\u003e mutation assay was performed according to previously described methods (Fukai et al., 2018). Briefly, \u003cem\u003eE. coli\u003c/em\u003e YG6020 was infected with the phage and spread on M9 salt plates containing chloramphenicol (Cm) and 6-thioguanine (6-TG). Plates were incubated for 72 h at 37\u0026deg;C to select colonies with a plasmid carrying the gene encoding chloramphenicol acetyltransferase, as well as mutated \u003cem\u003egpt\u003c/em\u003e. The 6-TG\u0026ndash;resistant isolates were cultured overnight at 37\u0026deg;C in LB broth containing 25 mg/mL of Cm, harvested by centrifugation (7,000 rpm, 10 min), and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Mutational spectra of 6-TG coding sequences were determined using PCR and direct sequencing, and a 739-bp DNA fragment containing \u003cem\u003egpt\u003c/em\u003e was amplified by PCR as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Sequence analysis was performed using Eurofins Genomics software (Tokyo, Japan)\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor the statistical comparison of the experimental and control groups in the \u003cem\u003egpt\u003c/em\u003e mutation assays, data were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations. The \u003cem\u003eF\u003c/em\u003e-test was initially performed to evaluate equality of variances. If variances were unequal, Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied. If not, Student\u0026rsquo;s \u003cem\u003et-\u003c/em\u003etest was used instead. In the case of the mutation spectrum analysis, \u003cem\u003eP\u003c/em\u003e-values were determined using Fisher\u0026rsquo;s exact test according to Carr and Gorelick [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In any case, \u003cem\u003ep\u003c/em\u003e-values lower than 0.05 were considered to indicate statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCytotoxicity of nanomaterials\u003c/h2\u003e\u003cp\u003eA 24-hour treatment with mesoporous silica resulted in a concentration-dependent decrease in cell viability, with significant growth inhibition observed at concentrations of 0.09, 0.13, and 0.25 mg/mL compared with the vehicle control (Fig.\u0026nbsp;1a). Exposure to graphene oxide showed no cytotoxicity at 0.1 mg/mL, with cell viability comparable to that of the control (Fig.\u0026nbsp;1b). However, cell viability decreased to 57% at 0.2 mg/mL exposure group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs control) and 70% in the 0.4 mg/mL exposure group (Fig.\u0026nbsp;1b). Although a clear concentration-dependent trend was not observed, cytotoxicity was observed in these exposure groups. Based on these results, the concentrations selected for the \u003cem\u003egpt\u003c/em\u003e mutation assay were 0.06 and 0.09 mg/mL for mesoporous silica, and 0.2 and 0.4 mg/mL for graphene oxide.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003emutagenicity of nanomaterials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003egpt\u003c/em\u003e mutation frequencies (MFs) in GDL1 cells exposed to mesoporous silica (0.06 and 0.09 mg/mL) increased compared with the control in a dose-dependent manner (Fig.\u0026nbsp;2a). The MF in the control group was 19.1 \u0026times; 10⁻\u003csup\u003e6\u003c/sup\u003e, whereas it was 16.9 \u0026times; 10⁻⁶ and 101 \u0026times; 10⁻⁶ in the 0.2 and 0.4 mg/mL of graphene oxide exposure groups, respectively, with a statistically significant increase observed at the higher concentration (Fig.\u0026nbsp;2b). The classes of \u003cem\u003egpt\u003c/em\u003e mutations induced by mesoporous silica are summarized in Table\u0026nbsp;1. Among the mutation profiles observed in the \u003cem\u003egpt\u003c/em\u003e coding sequence in GDL1 cells, the proportion of G:C to A:T transitions significantly increased from 14.3% in the control group to 24.6% in the mesoporous silica exposure group. In addition, exposure induced an increase in G:C to T:A transversions from 0% in the control group to 11.5%. Table\u0026nbsp;2 summarizes the classes of \u003cem\u003egpt\u003c/em\u003e mutations observed in GDL1 cells exposed to graphene oxide. At 0.4 mg/mL, both G:C to C:G transversions and G:C to A:T transitions were significantly elevated, with the frequency of G:C to C:G transversions increasing from 5.6% in the control group to 20.6% in the exposure group, and the proportion of G:C to A:T transitions increasing from 11.1% to 26.5%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe distribution of nanomaterial-induced and spontaneous mutations in the coding region of \u003cem\u003egpt\u003c/em\u003e is shown in Fig.\u0026nbsp;3. Of the 53 mutations induced by mesoporous silica, 10 were G to A transitions and 9 were T to G transversions. Mutation hotspots at positions 64, 164, and 416 were exclusively detected in the mesoporous silica-exposed group, while no such mutations were observed in the control group. Therefore, it is suggested that these mutations can be considered as mesoporous silica induced mutations. Transitions of C to T and transversion of G to C were only observed in the graphene oxide-treated group, therefore it is assumed that these mutations were induced by graphene oxide exposure. Notably, mutations at position 401 were detected exclusively in the graphene oxide group, indicating this site as a potential mutation hotspot.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe investigated the cytotoxicity and genotoxicity of mesoporous silica and graphene oxide using GDL1 cells. Cytotoxicity assays revealed a decrease in cell viability following exposure to both nanomaterials, indicating their cytotoxic potential. The cytotoxicity assay for graphene oxide did not show a clear concentration-dependent effect. This may be attributed to differences in the preparation methods of the exposure solutions: mesoporous silica was dispersed by ultrasonication, while graphene oxide was dispersed by pipetting. The lack of uniform dispersion in the high-dose graphene oxide group may have affected the results.\u003c/p\u003e\u003cp\u003eIn the \u003cem\u003ein vitro\u003c/em\u003e mutagenicity test, mesoporous silica significantly increased G:C to T:A and G:C to A:T base substitution mutations. Graphene oxide exposure led to a significant increase in G:C to A:T and G:C to C:G base substitution mutations. One of the known mechanisms through which nanosized particles exert adverse health effects is their ability to generate reactive oxygen species (ROS). Several studies have indicated that the genotoxicity of fine particulate matter, including nanomaterials, is linked to ROS production [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For instance, exposure to mesoporous silica has been shown to induce ROS generation and oxidative stress in BEAS-2B cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. ROS are known to cause oxidative modifications of DNA bases. Among the four DNA bases, guanine is particularly susceptible to oxidation, leading to the formation of 8-oxo-7,8-dihydroguanine (8-oxoguanine). During DNA replication, 8-oxoguanine can mispair with adenine, resulting in G:C to T:A transversions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, the increase in G:C to T:A transversions caused by mesoporous silica may be attributed to ROS generation.\u003c/p\u003e\u003cp\u003eIn general, the G:C to C:G transversion is thought to be a rare event in both spontaneous and chemically induced mutations. G:C to C:G transversions are generally considered rare events in both spontaneous and chemically induced mutations. However, Kato \u003cem\u003eet al\u003c/em\u003e. reported a significant increase in G:C to C:G transversions in \u003cem\u003egpt\u003c/em\u003e delta transgenic mice exposed to multi-walled carbon nanotubes (MWCNTs), a type of nanocarbon material similar to graphene oxide [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similar increases in G:C to C:G transversions have also been observed with other nano/microparticles, such as fullerenes (C60), carbon black (CB), and kaolin. In \u003cem\u003ein vitro\u003c/em\u003e assay systems, various oxidative stresses caused by sunlight, UV radiation, hydrogen peroxide and peroxyl radicals have frequently been reported to induce G:C to C:G transversions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Graphene oxide has also been shown to induce oxidative stress in A549 cells by generating ROS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. While 8-oxo-dG is a common form of DNA damage from oxidative stress, it does not cause G to C transversions because dG is not incorporated opposite 8-oxo-dG [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Instead, other oxidative guanine lesion products\u0026mdash;such as imidazolone (Iz), oxazolone (Oz), spiroiminodihydantoin (Sp), and guanidinohydantoin (Gh)\u0026mdash;have been identified [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Among these, Oz, Sp, and Gh are considered key contributors to G to C transversions via translational synthesis mechanisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, the G:C to C:G transversions induced by graphene oxide may involve the formation of these oxidized guanine derivatives.\u003c/p\u003e\u003cp\u003eIn this study, G:C to A:T transition mutations were also increased following treatment with both nanoparticles. G to A transitions are commonly observed in both spontaneous and chemically induced mutations and are often associated with deamination of 5-methylcytosine or alkylation of guanine [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Nitric oxide (NO) causes DNA damage by producing dinitrogen trioxide, which generates diazonium ions that induce DNA deamination and the formation of 8-nitro-dG. This compound can create apurinic sites, preferentially leading to G:C to A:T transition mutations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In a previous study, the lungs of rats exposed to mesoporous silica nanoparticles showed a significant increase in NO levels compared to control rats [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, graphene oxide has been reported to cause acute lung injury, inflammation, cell apoptosis, and DNA damage through the overproduction of ROS [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Graphene oxide exposure also significantly increased ROS production in mouse embryonic fibroblasts [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These findings suggest that graphene oxide may trigger inflammatory responses and increase NO production. Therefore, both mesoporous silica and graphene oxide may induce G:C to A:T transition mutations via NO generation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe demonstrated that mesoporous silica and graphene oxide exhibit cytotoxic and genotoxic potential in an in vitro assay using GDL1 cells, which were established from \u003cem\u003egpt\u003c/em\u003e delta transgenic mice. Based on the prominent mutation spectra observed, it is suggested that oxidative DNA damage and inflammation may be commonly involved in their mutagenicity. However, the detailed mechanisms underlying the genotoxicity of mesoporous silica and graphene oxide were not explored in the present study. Therefore, further investigations are required to elucidate the mechanisms of genotoxicity induced by these materials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by the Japan Agency for Medical Research and Development (AMED), Research on Regulatory Science of Pharmaceuticals and Medical Devices (Grant Number: JP22mk0101247).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.O. and Y.T. designed the study, A.O. acquired funding, R.I. performed the experiments, K.F. and S.H. analyzed the data, and R.I. wrote the initial draft. A.O. and Y.T. reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKohl Y, et al. Genotoxicity of nanomaterials: advanced in vitro models and high-throughput methods for human hazard assessment\u0026mdash;a review. Nanomaterials (Basel). 2020;10:1911.\u003c/li\u003e\n\u003cli\u003eGupta A, Kushwaha SS, Mishra A. A review on recent technologies and patents on silica nanoparticles for cancer treatment and diagnosis. Recent Pat Drug Deliv Formul. 2020;14:126\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eNarayan R, Nayak UY, Raichur AM, Garg S. Mesoporous silica nanoparticles: a comprehensive review on synthesis and recent advances. 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Nanotoxicology. 2013;7:452\u0026ndash;61.\u003c/li\u003e\n\u003cli\u003eTotsuka Y, Ishino K, Kato T, et al. Magnetite nanoparticles induce genotoxicity in the lung of mice via inflammatory response. Nanomaterials (Basel). 2014;4:175\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eTakeiri A, Mishima M, Tanaka K, Shioda A, Harada A, Watanabe K, et al. A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C. Mutat Res. 2006;609:102\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eFukai E, Sato H, Watanabe M, Nakae D, Totsuka Y. Establishment of an in vivo simulating co-culture assay platform for genotoxicity of multi-walled carbon nanotubes. Cancer Sci. 2018;109:1024\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eCarr GJ, Gorelick NJ. Mutational spectra in transgenic animal research: data analysis and study design based upon the mutant or mutation frequency. 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Austin (TX), New York (NY): Landes Bioscience/Springer; 2007. p.40\u0026ndash;53.\u003c/li\u003e\n\u003cli\u003eValentine MR, Rodriguez H, Termini J. Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG or abasic site formation. Biochemistry. 1998;37:7030\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eShin CY, Ponomareva ON, Connolly L, Turker MS. A mouse kidney cell line with a G:C\u0026rarr;C:G transversion mutator phenotype. Mutat Res. 2002;503:69\u0026ndash;76.\u003c/li\u003e\n\u003cli\u003ede la Parra S, Fern\u0026aacute;ndez Pamp\u0026iacute;n N, Garroni S, Poddighe M, de la Fuente Vivas D, Barros R, et al. Comparative toxicological analysis of two pristine carbon nanomaterials (graphene oxide and aminated graphene oxide) and their corresponding degraded forms using human in vitro models. Toxicology. 2024;504:153783.\u003c/li\u003e\n\u003cli\u003eShibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature. 1991;349:431\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eKino K, Sugiyama H. UVR-induced G:C to C:G transversions from oxidative DNA damage. Mutat Res. 2005;571:33\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eKino K, Sugiyama H. Possible cause of G:C\u0026rarr;C:G transversion mutation by guanine oxidation product, imidazolone. Chem Biol. 2001;8:369\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eShen JC, Rideout WM 3rd, Jones PA. The rate of hydrolytic deamination of 5-methylcytosine in double-stranded DNA. Nucleic Acids Res. 1994;22:972\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eSwann PF. Why do O6-alkylguanine and O4-alkylthymine miscode? The relationship between the structure of DNA containing O6-alkylguanine and O4-alkylthymine and the mutagenic properties of these bases. Mutat Res. 1990;233:81\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eHiraku Y, Guo F, Ma N, et al. 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Deciphering the underlying mechanisms of oxidation-state dependent cytotoxicity of graphene oxide on mammalian cells. Toxicol Lett. 2015;237:61\u0026ndash;71.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"genv","sideBox":"Learn more about [Genes and Environment](http://genesenvironment.biomedcentral.com/)","snPcode":"41021","submissionUrl":"https://submission.springernature.com/new-submission/41021/3","title":"Genes and Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mesoporous silica, Graphene oxide, Organoid, Genotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-7684406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7684406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNanomaterials such as mesoporous silica and graphene oxide are increasingly used in industrial, medical, and cosmetic applications due to their unique physical and chemical properties. However, their potential genotoxicity remains poorly understood. To evaluate the associated health risks of mesoporous silica and graphene oxide, we assessed their cytotoxicity and genotoxicity in GDL1 cells using trypan blue exclusion and \u003cem\u003egpt\u003c/em\u003e mutation assays, followed by mutation frequency and spectrum analysis through \u003cem\u003egpt\u003c/em\u003e gene sequencing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eA 24-hour exposure of mesoporous silica to GDL1 cells induced dose-dependent reductions in cell viability, as well as dose-dependent increases in \u003cem\u003egpt\u003c/em\u003e mutation frequencies at 0.06 and 0.09 mg/mL. Graphene oxide induced cytotoxicity at higher concentrations (0.2 and 0.4 mg/mL) and significantly increased \u003cem\u003egpt\u003c/em\u003e mutation frequency in the highest concentration exposure group compared to controls. Mutation spectrum analysis revealed a significant increase in G:C to A:T transitions in both the exposed groups. In addition, exposure to mesoporous silica significantly increased G:C to T:A transversions, while graphene oxide exposure significantly increased G:C to C:G transversions. Mutation hotspots at positions 64, 164, and 416 were identified exclusively in the mesoporous silica-treated group, indicating material-specific mutagenesis. Mutation hotspot at position 401 was detected exclusively in the graphene oxide group, indicating this site as a potential mutation hotspot.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese results demonstrate that both mesoporous silica and graphene oxide exhibit cytotoxic and genotoxic potential \u003cem\u003ein vitro\u003c/em\u003e. The mutation patterns suggest that oxidative DNA damage and inflammation may contribute to the observed genotoxicity. Further investigations are necessary to elucidate the molecular mechanisms underlying the mutagenicity of these nanomaterials and their implications for human health risk assessment.\u003c/p\u003e","manuscriptTitle":"Genotoxicity Assessment of Mesoporous Silica and Graphene Oxide in GDL1 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 20:50:57","doi":"10.21203/rs.3.rs-7684406/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-29T11:55:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T02:11:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T10:48:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141058261119141396555205807551142759549","date":"2025-10-07T14:37:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45938992148532491521162707026504426739","date":"2025-10-06T02:05:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-01T00:52:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-30T14:08:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-30T14:07:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes and Environment","date":"2025-09-22T12:05:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"genv","sideBox":"Learn more about [Genes and Environment](http://genesenvironment.biomedcentral.com/)","snPcode":"41021","submissionUrl":"https://submission.springernature.com/new-submission/41021/3","title":"Genes and Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"00d7238f-713c-4604-b915-5f4f8f5b5d95","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T15:59:57+00:00","versionOfRecord":{"articleIdentity":"rs-7684406","link":"https://doi.org/10.1186/s41021-025-00350-y","journal":{"identity":"genes-and-environment","isVorOnly":false,"title":"Genes and Environment"},"publishedOn":"2025-12-23 15:57:04","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-10-14 20:50:57","video":"","vorDoi":"10.1186/s41021-025-00350-y","vorDoiUrl":"https://doi.org/10.1186/s41021-025-00350-y","workflowStages":[]},"version":"v1","identity":"rs-7684406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7684406","identity":"rs-7684406","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}