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Mice in the control group were given saline solution by gavage. IVIS Spectrum imaging showed that the levels of autofluorescence intensity differed among the tissues (Table 1). Blood had the lowest autofluorescent signal. By comparison, epididymis had the highest autofluorescence intensity (123 times that of blood). Due to the autofluorescence, the folder increase (PS-treated group vs. control group) was used to indicate the change in strength. Because no significant differences existed among the different time points for each tissue in the control group, mean value of different time points was calculated as mean radiant efficiency of tissue in the control group to increase the stability of the autofluorescence intensity.
Table 1. Autofluorescence intensity of tissues in control group at different time points (unit: [(p/sec)/(μW/cm2)]/g, 1.00 x 108)
Tissues Mean ± SD 0.5 h (n = 3) 1 h (n = 3) 2 h (n = 3) 4 h (n = 3) Total Epididymis 138.0 ± 91.5 106.0 ± 21.5 97.2 ± 14.7 81.9 ± 10.40 106.0 ± 46.1 Subcutaneous fat 93.4 ± 27.8 83.3 ± 15.8 64.0 ± 14.4 70.1 ± 16.3 77.7 ± 20.4 Testis 62.1 ± 10.1 69.4 ± 2.5 61.4 ± 4.9 70.2 ± 11.7 65.8 ± 8.16 Perirenal fat 47.6 ± 28.7 48.0 ± 19.2 44.9 ± 13.7 44.8 ± 16.0 46.3 ± 17.3 Peritesticular fat 39.4 ± 3.4 46.8 ± 24.9 50.2 ± 12.5 41.9 ± 12.8 44.6 ± 13.9 Cerebellum 16.7 ± 2.6 16.8 ± 2.5 21.8 ± 0.8 16.9 ± 2.8 18.1 ± 3.0 Cerebrum 18.0 ± 0.70 18.4 ± 0.4 17.3 ± 3.34 17.64 ± 3.4 17.8 ± 2.1 Blood* 0.97 ± 0.28 0.78 ± 0.24 0.88 ± 0.03 0.78 ± 0.03 0.86 ± 0.18 Note. *Adjusted by volume (mL). -
Figure 1A shows the time course changes of fluorescence intensity in blood after administering PS beads by gavage at a dose of 200 mg∙kg-1∙BW. In the 100 nm PS exposure group, the fluorescence intensity increased significantly at 0.5, 1, and 2 h (P < 0.05) in comparison to the control group; the highest level was at t = 0.5 h. Up to 4 h, the fluorescence intensity decreased to the same level as the control group and was significantly lower than level at 0.5 h (P < 0.05). By contrast, no changes were observed for 3 and 10 μm PS exposure.
Figure 1. Time course changes of fluorescence intensity in blood, adipose tissues, nervous system, and reproductive system after administering PS beads by gavage at a dose of 200 mg∙kg−1∙BW. n = 3; (A) blood; (B) adipose tissues; (C) nervous system; (D) reproductive system. a, compared with control, P < 0.05; b, compared with 0.5 h, P < 0.05; c, compared with 1 h, P < 0.05; d, compared with 2 h, P < 0.05. PS, polystyrene
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For all adipose tissues examined, the increases in fluorescence intensity in the 100 nm PS-treated group were significant, even at the first time point (P < 0.05). However, the change trend was different among different adipose tissues. For subcutaneous and perirenal fat, fluorescence intensity continued to increase for 2 h (P < 0.05) after exposure, then decreased significantly at 4 h, but was still at a significantly high level in comparison to the control group (P < 0.05). The highest increasing level of fluorescence intensity was 3.65 times and 5.63 times as the control group, respectively. For peritesticular fat, the fluorescence intensity continued to increase during the entire time period (P < 0.05). At the 4 h time point, fluorescence intensity reached the highest level (4.41 times). In contrast, there were no significant changes observed in the 3 and 10 μm PS-treated groups (Figure 1B and Supplementary Figure S2-1A1−C3, available in www.besjournal.com).
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Cerebrum and cerebellum samples were detected by IVIS Spectrum. The results could reflect the ability of micro- and nano- PS beads to penetrate the blood-brain barrier. Figure 1C shows the time course changes of fluorescence intensity in the nervous system at different time points after PS beads exposure. After gavage at a dose of 200 mg∙kg−1∙BW, the fluorescence intensity of the cerebrum increased significantly at all time points (P < 0.05) in the 100 nm PS-treated group. With respect to the cerebellum, a significant increase in fluorescence intensity was also observed since the 1 h time point (P < 0.05). In the 3 μm PS-treated group, a significant increase in fluorescence intensity (1.54 times) was observed in the cerebellum 4 h after oral exposure (P < 0.05). In the 10 μm PS-treated group, no change was observed. The IVIS Spectrum small-animal imaging system images are shown in Supplementary Figure S2-2D1−E3.
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Figure 1D shows the time course changes of fluorescence intensity in the reproductive system at different time points after PS beads exposure. In the testis, the increase in fluorescence intensity at 4 h was significant (fold = 1.19, P < 0.05) in the 100 nm PS-treated group; however, no significance existed in the epididymis even if the level reached a 1.22 fold increase. Interestingly, the autofluorescence intensity of the epididymis was nearly 2 times the level of the testis. In the 3 μm PS-treated group, a significant increase in fluorescence intensity also reached statistical significance (P = 0.047). No significant changes were demonstrated for the 10 μm PS beads. The images in IVIS Spectrum are shown in Supplementary Figure S2-2F1−G3.
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In this study, the absorption of PS beads in blood was confirmed by CLSM and results are shown in Figure 2. Since IVIS Spectrum revealed that blood sample at 0.5 h have the strongest fluorescent intensity in the 100 nm PS-treated group, the blood samples at 0.5 h was further examined by CLSM. Even if green PS beads was found (Figure 2C–D), it was difficult to pinpoint the exact location in blood stream due to its small size. As for the 3 μm PS-treated group, the blood samples were also examined by CLSM because significant increase were found in cerebrum and testis by IVIS spectrum at 4 h after exposure. Interesting, the presence of PS bead was found since 2 h after exposure (Figure 2E–F). No any green fluorescence beads was found in saline control group (Figure 2A–B).
Figure 2. The evidence of PS beads in blood samples examined by CLSM and TEM. (A–B) are the fluorescence and bright-field images in the control group (600×). (C–D) are the images in 100 nm PS-treated group (600×). (E–F) are the images in 3 µm PS-treated group (600×). (G) (40,000×) is the TEM image for 100 nm PS bead. (H) (5,000×) is the TEM image for 3 µm PS bead. PS, polystyrene
Examination by TEM was also performed because the CLSM is not enough to distinguish the nanoparticle from fluorescent dye. Particles with diameter around 100 nm and 3 μm were found (Figure 2G–H).
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For solid samples, histopathology examination was conducted to confirm the significant findings revealed by IVIS spectrum. Thus, samples of subcutaneous fat, perirenal fat, peritesticular fat, cerebrum, cerebellum, and testis at 4 h in 100 nm PS treated group and samples of cerebellum and testis at 4 h in 3 μm PS-treated group were examined. Among all adipose tissues in 100 nm PS-treated group, fluorescence signals were brighter in adipose cells except lipid droplet (Figure 3). In the cerebrum, brighter fluorescence signals were found in blood vessels, perivascular space and nerve cells in cortex in 100 nm PS-treated group (Figure 4A2−A4). Furthermore, nerve cells tended to be hyperchromatic nuclei, pyknotic and irregular. Histological results of the cerebellum are shown in Figure 4C1−E4. The fluorescence signals were observed through the whole granular layer in 100 nm PS-treated group. The stronger signals in medulla were also found in 3 μm PS-treated group. Histological results of testis are shown in Figure 4F1−H4). The brighter fluorescence signals were found in the connective tissues and leydig cells, not the seminiferous tubule, in both 100 nm and 3 μm PS-treated groups. However, the thinner seminiferous tubule with reduced spermatogenic cell layers was observed in comparison to control group and the histopathological changes tended to be serious in 3 μm PS-treated group.
Figure 3. Histological examination of adipose tissues (200×). The first column is the image of HE staining. The second is the merge of both PS fluorescence and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of PS beads (green). PS, polystyrene. DAPI, 4',6-diaminidine-2-phenylindole.
Figure 4. Histological results of the cerebrum tissues, cerebellum tissues and testis tissues (200×). The first column is the image of HE staining. The second is the merge of both PS fluorescence and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of PS beads (green). PS, polystyrene. DAPI, 4',6-diaminidine-2-phenylindole.
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The results of leaching experiment examined by CLSM are shown in Figure 5. Different dispersing condition before exposure were revealed to directly affect the leaching. After dispersing PS suspensions by sonicating for 30 min as reported by Schür C et al. [28], the strong fluorescent signal pertaining to leaching was also observed (Figure 5E–F). In contrast, after dispersing PS suspensions by reversing gently as conducted in this study, the leaching was not observed except the residual particles that was not washed off and adhered to the silicone rubber strip, especially the edges (Figure 5C−D).
Figure 5. The leaching examination by confocal laser scanning microscope (100×). (A–B) are the fluorescence image and merged image (fluorescence and bright-field) of silicone rubber strip in 0.9% normal saline control group. (C–D) are the images in dispersing condition of reversing gently group. (E–F) are the images in dispersing condition of sonicating for 30 min group.
The confirmation was further conducted through TEM. Comparing between different dispersing conditions, the number of particles with smaller size observed frequently in group of sonicating for 30 min (Supplementary Figure S3B available in www.besjournal.com). Furthermore, the numbers of particles with diameter of > 80 nm, 50–80 nm and < 50 nm were counted for 3 view-fields in each group. The percentages of size > 80 nm in reversing gently group and sonicating for 30 min group were 92.3% and 65.6%; whereas the rates of size < 50 nm were 0 and 8.6% respectively.
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The results pertaining to the impact of pH on the properties of 3 μm PS beads are shown in Figure 6. CLSM examination revealed that there were no changes occurred to 3 μm PS beads before and after adding to the solution with a pH of 2.61, even for 4 h (Figure 6E), which indicated that low pH level was unable to affect MPs. The structures of 100 nm and 3 μm PS beads were observed by TEM (Figure 6F–G). For the 100 nm PS beads (95.37 nm), the thickness of dye-free surface was around 10 nm. In contrast, the thickness of dye-free surface was about 400 nm for 3 μm PS beads (2.60 μm).
Figure 6. CLSM examination for the impact of pH on 3 μm PS beads and TEM observation on 100 nm and 3 μm PS beads. (A) is the image before adding acid solution (600×). (B–E) are the images of 3 μm PS beads at 0.5, 1, 2, and 4 h after adding the equal volume of solution with a pH of 2.61 (600×). (F–G) are the TEM images of 100 nm (40,000×) and 3 μm (10,000×) PS beads. PS, polystyrene.
doi: 10.3967/bes2022.131
Evidence on Invasion of Blood, Adipose Tissues, Nervous System and Reproductive System of Mice After a Single Oral Exposure: Nanoplastics versus Microplastics
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Abstract:
Objective This study was designed to provide the evidences on the toxicokinetics of microplastics (MPs) and nanoplastics (NPs) in the bodies of mammals. Methods 100 nm, 3 μm, and 10 μm fluorescent polystyrene (PS) beads were administered to mice once by gavage at a dose of 200 mg/kg body weight. The levels and change of fluorescence intensity in samples of blood, subcutaneous fat, perirenal fat, peritesticular fat, cerebrum, cerebellum, testis, and epididymis were measured at 0.5, 1, 2, and 4 h after administration using an IVIS Spectrum small-animal imaging system. Histological examination, confocal laser scanning, and transmission electron microscope were performed to corroborate the findings. Results After confirming fluorescent dye leaching and impact of pH value, increased levels of fluorescence intensity in blood, all adipose tissues examined, cerebrum, cerebellum, and testis were measured in the 100 nm group, but not in the 3 and 10 μm groups except in the cerebellum and testis at 4 h for the 3 μm PS beads. The presence of PS beads was further corroborated. Conclusion After a single oral exposure, NPs are absorbed rapidly in the blood, accumulate in adipose tissues, and penetrate the blood-brain/testis barriers. As expected, the toxicokinetics of MPs is significantly size-dependent in mammals. -
Key words:
- Adipose accumulation /
- Blood-brain barrier /
- Blood-testis barrier /
- Blood uptake /
- Microplastics /
- Nanoplastics
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Figure 1. Time course changes of fluorescence intensity in blood, adipose tissues, nervous system, and reproductive system after administering PS beads by gavage at a dose of 200 mg∙kg−1∙BW. n = 3; (A) blood; (B) adipose tissues; (C) nervous system; (D) reproductive system. a, compared with control, P < 0.05; b, compared with 0.5 h, P < 0.05; c, compared with 1 h, P < 0.05; d, compared with 2 h, P < 0.05. PS, polystyrene
Figure 2. The evidence of PS beads in blood samples examined by CLSM and TEM. (A–B) are the fluorescence and bright-field images in the control group (600×). (C–D) are the images in 100 nm PS-treated group (600×). (E–F) are the images in 3 µm PS-treated group (600×). (G) (40,000×) is the TEM image for 100 nm PS bead. (H) (5,000×) is the TEM image for 3 µm PS bead. PS, polystyrene
Figure 3. Histological examination of adipose tissues (200×). The first column is the image of HE staining. The second is the merge of both PS fluorescence and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of PS beads (green). PS, polystyrene. DAPI, 4',6-diaminidine-2-phenylindole.
Figure 4. Histological results of the cerebrum tissues, cerebellum tissues and testis tissues (200×). The first column is the image of HE staining. The second is the merge of both PS fluorescence and DAPI dye. The third is the fluorescence image of DAPI dye. The fourth is the fluorescence image of PS beads (green). PS, polystyrene. DAPI, 4',6-diaminidine-2-phenylindole.
Figure 5. The leaching examination by confocal laser scanning microscope (100×). (A–B) are the fluorescence image and merged image (fluorescence and bright-field) of silicone rubber strip in 0.9% normal saline control group. (C–D) are the images in dispersing condition of reversing gently group. (E–F) are the images in dispersing condition of sonicating for 30 min group.
Figure 6. CLSM examination for the impact of pH on 3 μm PS beads and TEM observation on 100 nm and 3 μm PS beads. (A) is the image before adding acid solution (600×). (B–E) are the images of 3 μm PS beads at 0.5, 1, 2, and 4 h after adding the equal volume of solution with a pH of 2.61 (600×). (F–G) are the TEM images of 100 nm (40,000×) and 3 μm (10,000×) PS beads. PS, polystyrene.
Table 1. Autofluorescence intensity of tissues in control group at different time points (unit: [(p/sec)/(μW/cm2)]/g, 1.00 x 108)
Tissues Mean ± SD 0.5 h (n = 3) 1 h (n = 3) 2 h (n = 3) 4 h (n = 3) Total Epididymis 138.0 ± 91.5 106.0 ± 21.5 97.2 ± 14.7 81.9 ± 10.40 106.0 ± 46.1 Subcutaneous fat 93.4 ± 27.8 83.3 ± 15.8 64.0 ± 14.4 70.1 ± 16.3 77.7 ± 20.4 Testis 62.1 ± 10.1 69.4 ± 2.5 61.4 ± 4.9 70.2 ± 11.7 65.8 ± 8.16 Perirenal fat 47.6 ± 28.7 48.0 ± 19.2 44.9 ± 13.7 44.8 ± 16.0 46.3 ± 17.3 Peritesticular fat 39.4 ± 3.4 46.8 ± 24.9 50.2 ± 12.5 41.9 ± 12.8 44.6 ± 13.9 Cerebellum 16.7 ± 2.6 16.8 ± 2.5 21.8 ± 0.8 16.9 ± 2.8 18.1 ± 3.0 Cerebrum 18.0 ± 0.70 18.4 ± 0.4 17.3 ± 3.34 17.64 ± 3.4 17.8 ± 2.1 Blood* 0.97 ± 0.28 0.78 ± 0.24 0.88 ± 0.03 0.78 ± 0.03 0.86 ± 0.18 Note. *Adjusted by volume (mL). -
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22006Supplementary Materials.pdf