Assessment of the carcinogenic potential of particulate matter generated from 3D printing devices in Balb/c 3T3-1-1 cells | Scientific Reports

Scientific Reports volume 14, Article number: 23981 (2024) Cite this article

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Recently, there have been reports of sarcoma occurring in a Korean science teachers who used a 3D printer with acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) filaments for educational purposes. However, limited toxicological research data on 3D printing make it challenging to confirm a causal relationship between 3D printing and cancer. Therefore, occupational accidents involving teachers who have developed sarcoma have not been officially recognized. To address this gap, we aimed to evaluate the carcinogenic potential of particulate matter produced from ABS and PLA filaments commonly used in 3D printing. We created a generator mimicking 3D printing to generate particulate matter, which was used as an experimental material. The collected particulate matter was exposed to an in vitro system to investigate genetic damage, effects on cell transformation, and changes in carcinogenesis-related genes. Various assays, such as the comet assay, cell transformation assays, microarray analysis, and glucose consumption measurement, were employed. Cytotoxicity tests performed to determine the exposure concentration for the comet assay showed that cell viability was 83.6, 62.6, 42.0, and 10.2% for ABS at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. PLA showed 91.7, 80.3, 65.1, and 60.8% viability at exposure concentrations of 50, 100, 200, and 400 µg/mL, respectively. Therefore, 50 µg/mL was set as the highest concentration for both ABS and PLA, and 25 and 12.5 µg/mL were set as the medium and low concentrations, respectively. The comet assay showed no changes in genetic damage caused by the particulate matter. Cytotoxicity results performed to establish exposure concentrations in the transformation assay showed that ABS showed cell viability of 88.0, 77.4, 84.7, and 85.5% at concentrations of 1.25, 2.5, 5, and 10 µg/mL, respectively, but few cells survived at concentrations above 20 µg/mL. PLA showed minimal cytotoxicity up to a concentration of 20 µg/ml. Therefore, in the cell transformation assay, a concentration of 10 µg/mL for ABS and 20 µg/mL for PLA was set as the highest exposure concentration, followed by medium and low exposure concentrations with a common ratio of 2. In cell transformation assays, only one transformed focus each was observed for both ABS and PLA particulate matter-exposed cells. The microarray assay revealed changes in gene expression, with a 41.7% change at 10 µg/mL for ABS and an 18.6% change at 20 µg/mL for PLA compared to the positive control group. Analysis of carcinogenesis-related gene expression changes on days 1, 7, and 25 of the promotion phase revealed that in cells exposed to 5 µg/mL of ABS, RBM3 gene expression increased by 3.66, 3.26, and 3.74 times, respectively, while MPP6 gene expression decreased by 0.33, 0.28, and 0.38 times, respectively, compared to the negative control group. Additionally, the measurement of glucose consumption showed that it increased in cells exposed to ABS and PLA particulate matter. Our findings suggest that the carcinogenic potential of ABS- and PLA-derived particulate matter in 3D printing cannot be completely ruled out. Therefore, further research in other test systems and analysis of additional parameters related to carcinogenesis, are deemed necessary to evaluate the carcinogenic risk of 3D printers using these materials.

A 3D printer is a device that creates a three-dimensional object by stacking materials, such as polymers, metals, and paper, in a layer-by-layer manner based on 3D graphic design data. The fused deposition modeling (FDM) method of stacking filaments in 3D printing is not only cost-effective but also user-friendly and durable. Consequently, 3D printers find widespread application, particularly in Korea, where they serve as a popular educational tool (20.5%), with the majority being of the FDM type.

In 3D printers, a variety of synthetic resins (plastics) and metals are used as layering materials. FDM-type printers predominantly use thermoplastic filaments, such as acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA), nylon, polycarbonate (PC), polyvinyl alcohol (PVA), high-impact polystyrene (HIPS), and thermoplastic polyurethane (TPU). Specifically, ABS and PLA are primarily used in 3D printers intended for personal and educational applications.

ABS is a thermoplastic resin composed of three types of monomers: acrylonitrile, butadiene, and styrene. It exhibits good toughness (plastic deformation ability), impact resistance, and high-temperature resistance. Additionally, it is stronger, more flexible, and has superior chemical resistance compared to PLA, rendering it suitable for manufacturing parts and prototypes requiring impact resistance. In contrast, PLA, derived from natural resources, such as corn starch, is biodegradable and thus environmentally friendly. Moreover, it is easier to use and more economical than ABS.

Although concerns were raised about health problems caused by nanoparticles and various organic solvents generated during the 3D printing process, the risks were initially expected to be minimal because of the intermittent use of 3D printers. However, the occurrence of sarcoma in three teachers who used FDM-type 3D printers over an extended duration contradicted these initial expectations1.

Toxicity data available to evaluate the hazards of 3D printing are currently limited. While some reports suggest that particles generated from ABS exhibit higher cytotoxicity compared to PLA particles, which is fundamental in toxicity research2, other studies have indicated the opposite, reporting higher cytotoxicity for PLA particles3. The respiratory toxicities of ABS and PLA particles appear to be relatively mild. For instance, the LC50 of ABS pyrolysis products ranges from 15.0 to 28.5 mg/L4, and it has been reported that exposing experimental animals to a concentration of 240 µg/m3 causes only temporary and minimal toxicity in the respiratory and systemic organs5.

Currently, there are reports indicating the generation of organic solvents during 3D printing, including suspected carcinogens, such as formaldehyde, benzene, ethylbenzene, and styrene; however, there is currently no available data concerning particulate matter. Therefore, this study represents an early-phase investigation into carcinogenicity using an in vitro cell system to explore carcinogenesis, which is characterized by initiation and promotion through various pathways.

ABS filament (white; Cubicon Inc. Seoul, Republic of Korea) and PLA filament (white; Cubicon) were heated to 260 °C and 220 °C, respectively, using a 3D printer to generate particles. The generated particulate matter was collected at a rate of 10 L/min using an electrical low-pressure impactor (ELPI; Dekati Ltd., Kangasala, Finland). The size distribution of particulate matter was measured using ELPI and the particles collected by setting aluminum foils (Ø 25 mm) at each stage of the impact analysis of ELPI were weighed and prepared in DMSO (Invitrogen, D12345) at a concentration of 40 mg/mL and stored frozen until use.

The Balb/c 3T3-1-1 (JCRB0601) cell line was purchased frozen from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Tokyo, Japan). The obtained cell line was washed once with 10 mL of M10F culture medium (MEM; Gibco, Carlsbad, CA, USA; cat. # 11095080), 10% FBS (ATCC 302020), and 100 unit/mL penicillin-streptomycin (CytivaHyclone, Marlborough, MA; cat. # SV30010). Subsequently, the cells were placed in a 75-cm2 flask containing 10 mL of M10F culture medium and cultured in a CO2 incubator at 37 °C until reaching a cell density of 70–80%. Cultured cells were prepared at a density of 5 × 105 cells/mL in M10F culture medium supplemented with 5% DMSO, aliquoted at 1 ml each, stored in liquid nitrogen, and utilized as needed. For subculturing stored cells, the supernatant was removed, washed once with calcium-magnesium-free PBS (Gibco, cat. # 10010-23) and treated with 1.5 mL of trypsin-EDTA (Gibco 10010-23) in a CO2 incubator for approximately 2 min. Subsequently, 10 mL of M10F medium was added, pipetted several times, placed in a 15-mL tube, and centrifuged at 1000 rpm for 5 min at 4 °C in a Combi S14R centrifuge (Hanil Scientific, Daejeon, Republic of Korea) to eliminate residual Trypsin-EDTA from the supernatant.

A comet assay was performed to confirm gene damage. For this assay, Balb/c 3T3-1-1 cells were cultured in M10F culture medium. d-Mannitol (Sigma M4125) was used as a negative control, while ethyl methanesulfonate (Sigma M0880) was used as a positive control.

Cytotoxicity analysis was initially conducted for each test substance to determine the concentration (IC20) at which cell viability was reduced by 20%. Cells cultured at a density of 1 × 105 cells/mL in M10F culture medium were dispensed into 100 µL per well of a 96-well plate and incubated in a CO2 incubator at 37 °C for 1 day, followed by treatment with control and test substances. For cytotoxicity analysis, cultured cells were exposed to control and test substances for 4 h. Subsequently, WST-8 (AssayGenie, Ireland; cat. # MAE0207-500) prepared with fresh culture medium was added, and the absorbance at 450 nm was measured. The cell viability was calculated as follows:

For the Comet assay, cells were exposed to the control and test substances for 4 h, washed once with calcium- and magnesium-free PBS, and detached using trypsin-EDTA. Single-cell suspensions were prepared in PBS at a density of 1 × 105 cells/mL and mixed with LMAgarose (Trevigen, Gaithersburg, MD, USA; cat. # 4250-050-02) at a ratio of 1:10. Subsequently, 60 µL of the mixture was applied to a comet slide (Trevigen 4250-050-03) and allowed to solidify. Cells attached to the comet slide were lysed for 40 min in lysis solution (Trevigen; cat. # 4250-050-01) at 4 °C, followed by treatment in alkaline solution (0.3 mM sodium hydroxide, 1 mM EDTA) for 30 min to unwind the DNA, and subsequent electrophoresis at 21 V for 30 min. After electrophoresis, comet slides were washed twice with distilled water, fixed in 70% ethanol for 5 min, and stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA; cat. # S11494). Stained cells were analyzed for % tail intensity using Leica-DM2500 LEDs (Incident Light Fluorescence Filter system N2.1) from Leica microsystems (Germany) and Comet Assay IV (version 4.3.2) analysis software from Perceptive Instruments (UK). For analysis, at least 100 cells per test group were examined.

Cell transformation assays were performed to investigate carcinogenic potential of the test substances. For these assays, cells cultured in DF5F medium [DMEM/F12 (Gibco, cat. # 11320033), 5% FBS, and 100 unit/mL penicillin-streptomycin] were used. DMSO was used as the negative control material, while MCA (Sigma-Aldrich 213942) and TPA (Sigma-Aldrich P8139) were employed as positive controls during the initiation and promotion phases, respectively.

To determine the concentration of the test substance for the cell transformation assay, a cytotoxicity test was conducted. Cells prepared at a density of 4000 cells/mL in DF5F culture medium were distributed to each well of a 96-well plate (100 µL), and the day on which culture was initiated in a CO2 incubator was designated as day 0. The initiation phase was from day 1 to day 4, and the promotion phase was from day 8 to day 11. Each test substance was exposed during the initiation period and the promotion period according to the schedule below. Following exposure to the test substance, cell viability was calculated using WST-8.

For the cell transformation assay, 400 cells were seeded into each well of a 96-well plate on day 0, as outlined in the schedule given below, and the culture was initiated in a CO2 incubator. Morphological changes in cells were observed on day 39 post-exposure to each control and test substance during the initiation phase from days 1 to 4 (a total of 3 days) and the promotion phase from days 8 to 21 (a total of 13 days).

Transformed cells were verified by fixing cultured cells with methanol (Merck; cat. # 1.06009.1011), staining them with 5% Giemsa solution (Muto Pure Chemicals; cat. # 1500-3), and observing the transformed foci.

A microarray analysis was conducted to analyze gene expression patterns during cell transformation. For this analysis, 8000 cells per well (with two wells for each test group) were seeded into a 6-well plate on day 0 and cultured in a CO2 incubator. Cultured cells were then exposed to the corresponding control and test substances during the initiation (days 1–4) and promotion phases (days 8–15).

Another microarray analysis was performed by harvesting cells on days 1 (Day 7) and 8 (Day 15) of the promotion phase and outsourcing the analysis to Microgen Co., Ltd., Seoul, Republic of Korea. The microarray was tested three times, and the results from representative tests were utilized. The analysis process involved isolating RNA (TRIzol™ Plus RNA Purification Kit) from cells harvested on days 1 (Day 8) and 7 (Day 14) of the promotion phase, followed by cDNA synthesis (GeneChip® WT PLUS Reagent Kit), mRNA amplification (GeneChip®WT PLUS Reagent Kit), and sscDNA synthesis (GeneChip®WT PLUS Reagent Kit). Subsequently, the DNA was fragmented and terminal labeling (GeneChip®WT PLUS Reagent Kit), hybridized (GeneChip® Hybridization Oven), washing and staining (GeneChip®Fluidics Station) for gene analysis. The resulting data were normalized using signal space transformation-robust multichip analysis (SST-RMA) included in Affymetrix® Power Tools (APT). This was followed by gene-level SST-RMA to identify differentially expressed genes (DEGs). Statistical differences were determined based on the fold change. To further identify biological processes involved in carcinogenesis, we analyzed Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the online software Database for Annotation, Visualization, and Integrated Discovery. They identified pathway network relationships for DEGs.

We analyzed a total of eight genes expressed during cell transformation, including HMGA1, HMGA2, MPP6, RBM3, ZWINT, RAN, WT1, and Aurora-A. These eight genes served as cancer-related marker genes that were expressed throughout the entire transformation period when Bhas 42 cells were treated with TPA6. Among these genes, HMGA1, HMGA2, MPP6, RBM3, and ZWINT were selected for their close association with cancer progression. Additionally, RAN, WT1, and Aurora-A were selected and analyzed as markers implicated in cancer development and malignant transformation in both humans and experimental animals6. The primers used for amplifying each gene are listed in Table 1.

Real-time RT-PCR analysis was initiated on day 0 by seeding 400 cells per well in a 96-well plate and culturing them in a CO2 incubator. During the initiation phase from day 1 to day 4 (a total of 3 days) and the production phase from day 8 to day 21 (a total of 13 days), the corresponding control and test substances were administered. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using the SYBR™ Green Fast Advanced Cells-to-CT™ Kit (Invitrogen, Carlsbad, CA, USA) on the 1st (day 8), 7th (day 14), and 28th (day 35) days of the promotion period. The cultured cells were washed once with PBS and then lysed to isolate RNA (treated with 50 µL of lysis solution for 5 min and then with 5 µL of stop solution). Subsequently, 10 µL of isolated RNA and 40 µL of RT Master were added to a PCR tube. After mixing, reverse transcription cDNA was synthesized (reaction at 37 °C for 30 min, reaction at 95 °C for 5 min). Gene expression levels were quantified by adding 50 µL of PCR cocktail and 10 µL of reverse transcription cDNA to a PCR tube and performing real-time RT-PCR (50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s.

Glucose consumption analysis involved seeding 400 cells per well in a 96-well plate on day 0 and culturing them in a CO2 incubator during the initiation phase from day 1 to day 4 and the promotion phase from day 8 to day 21. The corresponding control and test substances were administered. On days 21 and 25, phenol red-free DF5F culture medium (DMEM/F12 (Gibco 21041-025), 5% FBS, 100 unit/mL penicillin-streptomycin) was added, and cells were cultured for 4 days. Glucose consumption was calculated by measuring the amount of glucose added on Day 25 and the amount of glucose remaining in the culture supernatant on Day 29 using a glucose assay kit (Sigma-Aldrich GAG020).

The results of the cytotoxicity test to determine the concentration for the comet and cell transformation assays were plotted with the mean and standard error calculated. The Comet assay results were obtained by measuring the % tail DNA intensity value for more than 100 cells in each group and displaying the median value along with the top 25% and bottom 25% values in a graph. Differences between test groups were compared using the Kruskal–Wallis test. Transformed foci were verified using a one-tailed chi-square test. Data quality checks of microarray data included visually confirming the expression distribution for each sample using the percentile, median, 25% percentile, 75% percentile, maximum value, and minimum value. Sample reproducibility was assessed by determining the Pearson correlation coefficient. Changes in gene expression levels were expressed as multiples of expression levels compared to the negative control group. The results of the real-time RT-PCR were also expressed as multiples of expression levels compared to the negative control group. Glucose consumption was determined through one-way analysis of variance and confirmed by Student’s t-test for significance.

The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the particulate matter generated from the 3D printing device using ABS and PLA filaments were analyzed. For ABS, the MMAD was 0.156 μm with a GSD of 1.543, while for PLA, the MMAD was 0.173 μm with a GSD of 1.481 (Fig. 1).

Size distribution of printing particles for ABS and PLA filaments. Cumulative mass of ABS (A), mass size distribution of ABS (B), cumulative mass of PLA (C), mass size distribution of PLA (D). ABS acrylonitrile butadiene styrene, PLA polylactic acid.

After performing a cytotoxicity test to determine exposure concentrations for the comet assay, cell viability remained above 80% for mannitol, a negative control substance, up to 10 mM, and for ethyl methanesulfonate up to 5 mM (Fig. 2A). As the comet assay is recommended to be performed at cytotoxic concentrations below 20%, the concentrations of mannitol were set at 10 and 5 mM, while those of ethyl methanesulfonate were set at 5 and 2.5 mM. For ABS, the cell viability was 83.6% at a concentration of 50 µg/mL, while it dropped to 62.6%, 42.0%, and 10.2% at higher concentrations of 100, 200, and 400 µg/mL, respectively. ABS decreases cell viability in a concentration-dependent manner.

Cell viability for ABS and PLA particles for establishing exposure concentrations in the comet assay.

For PLA, the cell survival rates were 91.7%, 80.3%, 65.1%, and 60.8% at concentrations of 50, 100, 200, and 400 µg/mL (Fig. 2B). Based on these results, 50 µg/mL was set as the highest concentration for both ABS and PLA, with 25 and 12.5 µg/mL set as the medium and low concentrations, respectively.

When cells were electrophoresed for the comet assay, normal cells maintained a round shape (Fig. 3A), whereas those with genetic damage exhibited a comet shape with a tail (Fig. 3B). The Comet assay utilizes these characteristics to analyze genetic damage based on tail length or intensity in cell images. In this study, the median tail intensities were 2.15% and 3.05% after exposure to 5 and 10 mM mannitol, respectively, which met the conditions of the negative control group. Conversely, exposure to ethyl methanesulfonate resulted in significant differences compared to the negative control, with tail intensities of 18.03% at 1.25 mM and 40.51% at 2.5 mM, indicating compliance with the comet assay conditions (Fig. 3C,D). When exposed to the exposure concentrations of 12.5, 25, and 50 µg/mL of ABS and PLA, the median tail intensity of each exposure group was 1.15%, 0.64%, and 0.77% for ABS, and 0.42%, 0.78%, and 0.30% for PLA. In addition, no significant DNA damage occurred at any exposure concentration of ABS and PLA (Fig. 3E,F).

Assessment of DNA damage in individual cells following exposure to ABS and PLA substances. The images of the comet analysis were taken using the Comet Assay IV (Ver. 4.3.2) (Perceptive Instruments, UK) program https://www.instem.com/solutions/genetic-toxicology/comet-assay.php. Representative comet images of (A) mannitol and (B) ethyl methanesulfonate, results of (C) the negative control Mannitol and (D) positive control Ethyl methanesulfonate, and results of test substances (E) ABS and (F) PLA.

To determine the exposure concentration in the cell transformation assay, cytotoxicity was assessed by exposing the cells to the test substance during the initiation phase (days 1–4) and the promotion phase (days 8–11). For the cytotoxicity test, 0.1% DMSO was used as the negative control, while 1 µg/mL MCA was used as the positive control during the initiation phase and 50 ng/mL TPA during the promotion period. The cell survival rate with the negative control material was 106.1%, whereas with MCA or TPA treatment, the cell survival rate was 82.2% (Fig. 4A).

Cytotoxicity results for establishing exposure concentrations of ABS and PLA substances. (A) Cytotoxicity of negative and positive control substances, (B) cytotoxicity of test substances. DD DMSO treatment during the initiation and promotion phases, DT DMSO during the initiation phase and TPA treatment during the promotion phase, MD MCA treatment during the initiation phase and DMSO treatment during promotion phase, MT MCA treatment during the initiation phase and TPA treatment during the promotion phase.

Cytotoxicity tests of the test substances were conducted across a concentration range from 1.25 to 40 µg/mL. ABS exhibited cell survival rates of 88.0%, 77.4%, 84.7%, and 85.5% at concentrations of 1.25, 2.5, 5, and 10 µg/ml, respectively, all exceeding 80% survival rate. However, almost no surviving cells were observed at concentrations above 20 µg/mL. PLA showed minimal cytotoxicity across all treatment concentrations (Fig. 4B).

The results of the cell transformation assay for the negative and positive control materials are shown in Fig. 5. While no foci believed to have induced cell transformation were observed in the negative control treated with DMSO, foci of transformed cells were observed in 21 out of 96 wells in the positive control treated with MCA and TPA.

Morphological confirmation results of cell transformation following exposure to control substances. Image of the morphology of (A) normal cells and (B) transformed cells (magnification 32x); (C) Images of the 96-well plate of normal control cells and (D) positive control treated cells. Circles in image (D) indicate wells containing transformed cells.

ABS treatment at a concentration of 20 µg/mL resulted in some wells devoid of cells, with no cell transformation foci observed in those containing cells. Even at exposure concentrations of 2.5, 5, and 10 µg/mL, no transformed cells were detected. Conversely, when PLA was administered at concentrations of 5 and 10 µg/mL, one well out of 96 exhibited a focus of morphological change (Fig. 6B,C,E,F), yet no transformation focus was observed at concentrations of 2.5 and 20 µg/mL (Fig. 6A,D).

Morphological confirmation results of cell transformation following exposure to PLA. Images of the 96-well plate subjected to PLA treatment at concentrations of (A) 2.5 µg/mL, (B) 5 µg/mL, (C) 10 µg/mL, and (D) 20 µg/mL.

A total of 22,206 genes were identified by microarray analysis. Of these genes, 5041 had an absolute fold change (fc) of two or more compared to the negative control group. The results are shown in Supplementary Table S1, with 147 carcinogenic pathway genes were separately tabulated in Supplementary Tables S2 and S3.

Microarray analysis showed that in the positive control group, 1561 genes were over-expressed by more than 2-fold on day 1 and 796 on day 8 of promotion, and 448 genes were over-expressed on both days. Among the carcinogenic pathway genes, 55 were upregulated on day 1 of promotion, 24 on day 8, and 18 on both days (Fig. 7A). Conversely, there were 1318 genes with an fc value decrease of more than 2-fold on the 1st day of promotion and 534 on the 8th day, with 247 genes decreasing on both days. Among the carcinogenic pathway genes, 58 were downregulated on the 1st day of promotion, 23 on the 8th day, and 16 on both days (Fig. 7B). Tables 2 and 3 show the number of genes whose fc values changed more than 2-fold after ABS or PLA exposure compared to the negative control. The percentage of up- and down-regulated genes compared to the positive control was 21.6% at 5 µg/mL exposure concentration and 10.9% at 10 µg/mL exposure concentration on day 1 of promotion for ABS exposure, and 25.8% at 5 µg/mL exposure concentration and 34.1% at 10 µg/mL exposure concentration for ABS on day 8 of promotion. In the PLA-exposed group, the incidence was 5.0% at the 10 µg/mL exposure concentration and 20.3% at the 20 µg/mL concentration on day 1 of promotion, and 22.9% and 25.7% at the 10 and 20 µg/mL exposure concentrations, respectively, on day 8 of promotion (Table 2). In genes related to carcinogenic pathways, the ABS exposure group had 15.0% at 5 µg/mL exposure and 8.8% at 10 µg/mL on day 1 of promotion, and 8.5% and 31.9% on day 8 of promotion, respectively. In the PLA-exposed group, 2.7% at 10 µg/mL and 13.3% at 20 µg/mL on day 1 of promotion, and 6.4% and 17.0% at 10 and 20 µg/mL on day 8 of promotion, respectively (Table 3).

Number of genes with absolute fold-change (fc) values increased or decreased by more than 2-fold following exposure to positive control substances.

The Kyoto Encyclopedia for Genes and Genomics (KEGG) analysis results are presented in Table 4. The analysis revealed significant alterations in genes associated with crucial carcinogenic pathways, such as the vascular endothelial growth factor (VEGF), p53, cell cycle, estrogen, and mitogen-activated protein kinase (MAPK) signaling pathways, within the positive control group when compared to the negative control group. In contrast, minimal changes in gene expression within the carcinogenic pathways were observed following exposure to ABS and PLA.

To investigate changes in carcinogenesis-related genes in cells exposed to ABS and PLA, RNA was extracted and real-time RT-PCR was performed. To ensure the reproducibility of gene expression analysis, GAPDH was selected as an internal standard gene, and the experimental conditions were validated. The melting point of the GAPDH primer used was determined to be 87.9 °C, with an efficiency of 109.8%, indicating its suitability for analysis (Supplementary Fig. S1).

Changes in carcinogenesis-related genes following exposure to ABS and PLA are shown in Figs. 8 and 9. HMGA1 and HMGA2, non-histone proteins associated with various cancers and gene regulation7,8,9,10,11,12,13,14,15,16,17, exhibited 4.68- and 5.32-fold higher expression, respectively, in the positive control group than in the negative control group on the 1st day of promotion, and 2.49- and 2.32-fold higher expression, respectively, on the 8th day of promotion. By the 25th day of promotion, their expression levels increased by 1.32- and 2.51-fold, respectively. However, following exposure to ABS and PLA, no gene showed expression levels were more than twice or less than 0.5 times that of the negative control group (Fig. 8).

Expression ratio of HMGA1 and HMGA2 genes following substance exposure. (A,B) HMGA1, (C,D) HMGA2.

Expression ratio of RBM3 and MPP6 genes following substance exposure. (A,B) RBM3, (C,D) MPP6.

In this study, RBM3, a translation regulatory protein, showed no difference in expression level between the positive and negative control groups. However, when exposed to a concentration of 5 µg/ml ABS, its expression levels on days 1, 7, and 25 of the promotion phase were 3.66-, 3.26-, and 3.74-fold higher, respectively, compared to the negative control group.

MPP6, which is consistently expressed in glioblastoma stem cell cultures and inconsistently in neural stem cell cultures18, exhibited 0.33-, 0.28-, and 0.38-fold lower expression than the negative control group on days 1, 7, and 25 after promotion, respectively, at an exposure concentration of 5 µg/mL ABS (Fig. 9). Aurora-A, an oncogene that plays an essential role in regulating cell division during mitosis and promoting tumorigenesis in various types of cancers, including solid tumors and hematological malignancies19, exhibited a 2.22-fold higher expression in the positive control group than in the negative control group on day 7 of promotion. Recent studies have underscored its overexpression in majority of adult leukemias, including acute myeloid leukemia, chronic myeloid leukemia, and acute lymphoblastic leukemia, and in certain patients with myelodysplastic syndrome20,21,22,23,24,25,26,27,28.

The WT1 gene, which is overexpressed in breast cancer, testicular cancer, ovarian cancer, and melanoma29,30,31,32,33,34, showed no change in expression level exceeding 2-fold or falling below 0.5-fold in the positive control group compared to the negative control group. However, on the first day of promotion, we observed more than two-fold increase in expression level at the ABS concentration of 10 µg/mL and at the PLA concentrations of 10 and 20 µg/mL (Fig. 10).

Expression ratios of Aurora-A, WT1, RAN, and ZWINT genes following substance exposure. (A,B) Aurora-A; (C,D) WT1; (E,F) RAN; (G,H) ZWINT.

The RAN gene, which has been identified as having increased expression in a variety of cancers, including breast, kidney, stomach, colon, pancreas, ovarian, and lung cancers, as well as brain, bladder, adrenal, thyroid, esophageal, uterine, liver, liver, testicular, prostate, and cervical cancers35,36,37,38,39,40,41,42, and the ZWINT gene, which is a key regulatory protein of the mitotic checkpoint, regulates the cell cycle43, and is known to be associated with chromosomal instability44, showed no change in expression of more than 2-fold or less than 0.5-fold in any group compared to the negative control (Fig. 10E–H).

Glucose consumption was monitored for a period of 4 days from the 17th day (day 25) to the 12th day (day 29) of promotion. The positive control group exhibited a glucose consumption rate of 155.2% compared to the negative control group. Exposure to ABS at concentrations of 2.5, 5, and 10 µg/mL resulted in rates higher than the negative control group at 142.2%, 136.9%, and 142.2%, respectively. Similarly, exposure to PLA at concentrations of 5, 10, and 20 µg/mL led to glucose consumption rates of 142.5%, 128.0%, and 131.0%, respectively, compared to the negative control group (Fig. 11).

Results of analyzing glucose consumption following substance exposure. (A) ABS, (B) PLA.

Until sarcoma, a rare form of cancer was reported among science teachers in Korea who performed 3D printing work using plastic materials, it was vaguely thought that accidents due to 3D printers for simple home or educational use were unlikely to occur. There had been a general assumption that such activities posed little risk to human health. This incident prompted a reassessment of safety and health protocols in 3D printing work, leading to a shift towards to PLA, which is believed to be less harmful than ABS filaments.

Currently, the conditions surrounding the use of 3D printing necessitate data on potential human health hazards, especially regarding carcinogenicity. Recently, numerous studies have reported harmful substances emitted during 3D printing using plastics such as ABS and PLA. While these studies mainly focused on identifying the types and properties of materials released during 3D printing45,46,47, there are also studies that have delved into associated hazards48,49,50,51,52,53.

Previous studies have shown that ABS emissions increase reactive oxygen species (ROS) production, oxidative damage, cell necrosis and apoptosis, and inflammatory transmitters in cultured cells in a dose-dependent manner50. However, in this study, the effects associated with oxidative damage were not observed in the KEGG pathway and comet analysis. Additionally, ABS has been associated with transient and minimal toxicity to the overall body and respiratory system, acute hypertension, and microvascular dysfunction5,45. Epidemiological studies on 3D printing have highlighted potential respiratory effects53,54,55. In Korea, 3D printer-related carcinogenesis was first reported in science teachers using 3D printers by Wook et al.1. Choi et al. were unable to definitely ascertain a causal relationship between 3D printing and carcinogenesis in their epidemiological investigation. Essentially, the relationship between FDM 3D printing using plastic materials and carcinogenesis remains uncertain, with insufficient scientific evidence to establish causality.

Therefore, this study attempted to determine the carcinogenic potential of particulate matter generated from ABS and PLA filaments, common commercial plastics, using a 3D printing particle-generating device. To this end, microarrays were performed using 3T3 cells derived from BALB/c mice to evaluate changes in the expression profiles of cancer-related genes during cellular transformation. Additionally, glucose consumption was quantified to assess its potential modulation in response to ABS and PLA exposure.

ABS and PLA filaments were generated in a 3D printing generator at 260℃ and 220℃, respectively, in accordance with the recommended printing temperatures for each filament. The resulting particulate matter had an MMAD and a GSD of 0.156 μm and 1.543 for ABS and 0.173 μm and 1.481 for PLA, respectively. These ultrafine particles included nanoparticles and were similar to particles generated by 3D printing in previous studies51,52,53,54,55,56.

The comet assay has previously been used to study e.g. particles from selective laser melting (SLM) printers employing metallic materials57: however, particles from ABS and PLA materials yet to be tested using this technique. A recent study reported that particles emitted from PLA material induced DNA damage in SAECs58. Nonetheless, in this study, no increase in DNA damage was observed when particles were tested up to a concentration of 50 µg/mL.

A cell transformation assay was conducted to investigate the potential link between particulate matter generated from 3D printing and carcinogenesis. In this study, the negative control group treated with DMSO showed no cell transformation foci. However, in the positive control group treated with MCA in the initiation stage and TPA during the promotion stage, more than 20 cell-transformed foci were observed among the 96 wells, validating the test conditions. An exposure to ABS or PLA at a concentration of 10 µg/mL resulted in no significant effect on cell viability. However, at a concentration of 40 µg/mL, ABS exposure led to almost negligible cell viability, while PLA exposure caused no decrease in cell viability. Consequently, an additional test was performed, which revealed that the cell viability was 74.7% at an exposure concentration of 100 µg/mL PLA (data not shown). Therefore, ABS was deemed more cytotoxic than PLA. In the cell transformation assay of ABS and PLA, no cell transformation foci were observed at all exposure concentrations of ABS (2.5, 5, 10, and 20 µg/mL). At both 10 and 20 µg/mL PLA exposure concentrations, transformed foci were observed in only one well each. Thus, it was concluded that ABS did not induce cell transformation under the conditions of this assay. For PLA, one transformed focus was observed at each of the exposure concentrations of 10 and 20 µg/mL. However, in the positive control exposure group, transformed foci were observed in more than 20 wells. Therefore, PLA was also not evaluated as positive.

Research is underway to confirm the carcinogenic mechanism by measuring the expression levels of genes related to carcinogenesis or microarrays during cell transformation, aiming to enhance the ability of cell transformation tests in confirming carcinogenesis6,59,60,61. We also sought to confirm the carcinogenic potential of ABS and PLA using cell transformation assays. Herein, RNA was isolated on the 1st and 8th day of promotion, followed by microarray analysis. When MCA was used as a positive control in the initiation phase and TPA in the promotion phase, among 5041 genes, 1561 genes were upregulated on day 1 of promotion and 796 genes were upregulated on day 8. The number of downregulated genes was 1318 on day 1 and 247 on day 8 of promotion. These results align with findings from other studies using microarrays6,59. An analysis of 147 carcinogenesis-related genes revealed a significant change in the expression levels of all genes on the 1st day of promotion, similar to the overall gene expression pattern. Among them, 55 genes were upregulated and 58 were downregulated, potentially representing KEGG carcinogenesis pathways, as previously described.

Compared to the positive control group, the ABS-treated group exhibited 15.0% and 8.8% alteration in the expression of carcinogenesis-related genes on the first day of promotion when exposed to ABS concentrations of 5 and 10 µg/mL, respectively. Conversely, exposure to PLA at a concentration of 10 µg/mL resulted in a 2.7% difference compared to the positive control group, while at 20 µg/mL PLA, the difference was low at 13.3%. Hence, the carcinogenic potential was considered relatively low compared to the positive control group. However, when exposed to 10 µg/mL ABS, the percentage of up-regulated genes on the 8th day of promotion was 41.7% compared to the positive control group. Therefore, it was difficult to conclude that particles generated from ABS did not affect changes in genes related to carcinogenesis. The KEGG carcinogenic pathway analysis identified significant changes in genes associated with key carcinogenic pathways, including VEGF, p53, cell cycle, estrogen, and MAPK signaling pathways, in the positive control group. On the other hand, when exposed to particulate matter generated from ABS and PLA filaments, ABS showed significant changes in chemical carcinogenesis-reactive oxygen species at 5 µg/mL on day 1, transcriptional misregulation in cancer at 10 µg/mL, and transcriptional misregulation in cancer, chemical carcinogenesis-DNA adducts, and choline metabolism in cancer at 10 µg/mL on day 8. In the case of PLA, no pathway showed any significant changes related to carcinogenesis.

Additionally, changes in the expression levels of carcinogenesis-related genes were confirmed using the RT-qPCR method. Among several cancer-related genes examined in this study, HMGA1, HMGA2, Aurora-A, RBM3, MPP6, WT1, RAN, and ZWINT were selected. HMGA1 and HMGA2 regulate cell cycle and are known to promote microRNA-mediated cancer pathways62,63. They are highly expressed in various cancers, such as cervical and breast cancers64. The RBM3 gene is crucial for cell proliferation and has proto-oncogenic properties, aiding in cell survival in adverse environments. Additionally, it is highly expressed in various cancers, including colon cancer, testicular germ cell tumor, metastatic urothelial carcinoma, esophageal and gastric adenocarcinomas, and astrocytoma65. MPP6 is involved in cell adhesion and is overexpressed in liver cancer66, while Aurora-A plays a role in mitosis and is overexpressed in various cancers such as lung, rectal, and breast cancers67. WT1 promotes cell proliferation in leukemia and solid cancers, contributing to cancer cell invasion and metastasis52. RAN regulates molecular movement between the nucleus and cytoplasm and cell cycle progression; however, its expression is disrupted during metastasis in carcinogenesis68. ZWINT is overexpressed in liver, breast, and other cancers69,70. In this study, HMGA1 and HMGA2 showed 4.68- and 5.32-fold higher expression, respectively, on the 1st day of promotion in the positive control group than in the negative control group, followed by 2.49- and 2.32-fold higher expression, respectively, on the 8th day of promotion, and 1.32- and 2.51-fold higher expression, respectively, on the 25th day of promotion. These results were consistent with the microarray results, which showed the highest expression on the first day of promotion followed by a decrease over time. Notably, the overexpression of HMGA1 and HMGA2 was observed in the positive control group, but not from exposure to ABS and PLA particles. The RBM3 gene showed high expression at an exposure concentration of 5 µg/mL ABS, while the WT1 gene exhibited more than two times higher expression at an exposure concentration of 10 µg/mL ABS. Moreover, the WT1 gene showed elevated expression even at exposure concentrations of 10 and 20 µg/mL PLA. However, it may be premature to conclude that these alterations contribute to cancer, especially considering the morphological changes observed in the cell transformation analysis. In this study, microarray data and genetic changes were analyzed during the promotion phase. The first day of promotion corresponds to 8 days from the date of the initial exposure to the test substance if the initiation phase is included, while the 8th day of promotion corresponds to the 15th day. It is conceivable that measuring genetic changes during the initiation stage may induce alterations in other genes related to carcinogenesis.

Additionally, based on epidemiological studies and observations of tumor types in humans, changes in energy metabolism have been implicated in malignant cell transformation71. Therefore, in this study, energy metabolism, particularly glucose consumption, was assessed in a cell transformation assay. Elevated glucose consumption was observed in both the positive control group and the groups treated with test substances compared to the negative control group. For the positive control group, these findings indicate transformed cell proliferation; however, if it is considered unrelated to cell proliferation in the test substance-exposed group, further investigation is warranted.

Based on the aforementioned findings, it appears challenging to definitively assert a causal relationship between particles generated from ABS and PLA filaments in 3D printers and carcinogenesis, considering their effects on DNA damage, cell transformation, carcinogenesis-related gene expression, and glucose consumption in this study. However, observation of transformed foci in cells exposed to PLA concentrations of 5 and 10 µg/mL, changes in the expression levels of certain oncogenesis-related genes, such as RBM3 and WT1, as confirmed through RT-qPCR analysis, and the increase in glucose consumption during the cell transformation assay, suggest a need for further investigation. Thus, ruling out any potential association with carcinogenesis proves challenging. To our knowledge, this study is one of the first attempts to investigate the carcinogenic potential of particulate matter generated from 3D printers using ABS and PLA filaments. Comparative studies involving comet and cell transformation assays have remained inconclusive until recently.

Although this study could not clearly demonstrate the cell transformation potential of particulate matter generated by a 3D printer using ABS and PLA filaments, carcinogenicity could not be ruled out completely. Therefore, it is concluded that further research is necessary to obtain additional evidence for a more comprehensive evaluation of the carcinogenic effects of 3D printing using ABS and PLA filaments. Additional studies could involve test systems, such as human cell-derived organoids or in vivo cancer induction, better representing the effects in humans compared to in vitro cell culture systems. Alternatively, studies could utilize test systems simulating 3D printing operations, such as a mixing system of particulate matter and organic solvent generated from a 3D printer. that better mimic 3D printing operations. Regardless of the chosen test system, the analysis of additional carcinogenesis-related parameters will be crucial to obtain additional evidence supporting the correlation between 3D printing using ABS and PLA filaments and sarcoma.

In conclusion, our data represent the initial evidence for assessing the carcinogenic potential of particulate matter generated during 3D printing with ABS and PLA filaments, based on carcinogenic mechanisms. Considering our study findings, we cannot definitively conclude that our results are entirely unrelated to the causal relationship with carcinogenesis.

Data collected and analyzed during the study will be made available by the corresponding author upon reasonable request.

Fused deposition modeling

Acrylonitrile-butadiene-styrene

Polylactic acid

Polycarbonate

Polyvinyl alcohol

High-impact polystyrene

Thermoplastics polyurathane

Electrical low-pressure impactor

Dimethyl sulfoxide

Minimum essential medium

Fetal bovine serum

Phosphate buffered saline

Water-soluble tetrazolium-8

Dulbecco’s modified eagle medium/ham’s F12

3-methyl cholanthrene

12-O-tetradecanoyl phorbol-13-acetate

Complementary deoxyribo nucleic acid

Messenger RiboNucleic acid

Reverse transcription polymerase chain reaction

Mass median aerodynamic diameter

Geometric standard deviation

High mobility group A1

High mobility group A2

Membrane protein, palmitoylated 6

RNA binding motif protein 3

Zeste white 10 interactor

RAs-related nuclear protein

Wilms’ tumor 1

Glyceraldehyde-3-phosphate dehydrogenase

Kyoto encyclopedia of genes and genomes

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This study was conducted as an independent research project at Korea Occupational Safety and Health Research Institute. We would like to thank Dr. SungBae Lee for creating the 3D printer generator used in this study.

This work was supported by the Korea Occupational Safety and Health Research Institute “Chemical Research Fund (2023)”.

Inhalation Toxicity Research Center, Occupational Safety and Health Research Institute, Korea Occupational Safety and Health Agency, 30, Expro-ro 339 beon-gil, Yuseong-gu, Daejeon, Republic of Korea

CheolHong Lim & DongSeok Seo

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CHL and SDS designed the research project, performed the experiments, collected and analyzed the data, and wrote the manuscript.

Correspondence to DongSeok Seo.

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Lim, C., Seo, D. Assessment of the carcinogenic potential of particulate matter generated from 3D printing devices in Balb/c 3T3-1-1 cells. Sci Rep 14, 23981 (2024). https://doi.org/10.1038/s41598-024-75491-1

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DOI: https://doi.org/10.1038/s41598-024-75491-1

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