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. 2023 Feb 12;12(2):464.
doi: 10.3390/antiox12020464.

Oxidative Stress in Long-Term Exposure to Multi-Walled Carbon Nanotubes in Male Rats

Ewa Florek  1 , Marta Witkowska  2   3 , Marta Szukalska  1 , Magdalena Richter  4 , Tomasz Trzeciak  4 , Izabela Miechowicz  5 , Andrzej Marszałek  6 , Wojciech Piekoszewski  7 , Zuzanna Wyrwa  1 , Michael Giersig  3   8
Affiliations

Oxidative Stress in Long-Term Exposure to Multi-Walled Carbon Nanotubes in Male Rats

Ewa Florek et al. Antioxidants (Basel). .

Abstract

Multi-walled carbon nanotubes (MWCNTs) serve as nanoparticles due to their size, and for that reason, when in contact with the biological system, they can have toxic effects. One of the main mechanisms responsible for nanotoxicity is oxidative stress resulting from the production of intracellular reactive oxygen species (ROS). Therefore, oxidative stress biomarkers are important tools for assessing MWCNTs toxicity. The aim of this study was to evaluate the oxidative stress of multi-walled carbon nanotubes in male rats. Our animal model studies of MWCNTs (diameter ~15-30 nm, length ~15-20 μm) include measurement of oxidative stress parameters in the body fluid and tissues of animals after long-term exposure. Rattus Norvegicus/Wistar male rats were administrated a single injection to the knee joint at three concentrations: 0.03 mg/mL, 0.25 mg/mL, and 0.5 mg/mL. The rats were euthanized 12 and 18 months post-exposure by drawing blood from the heart, and their liver and kidney tissues were removed. To evaluate toxicity, the enzymatic activity of total protein (TP), reduced glutathione (GSH), glutathione S-transferase (GST), thiobarbituric acid reactive substances (TBARS), Trolox equivalent antioxidant capacity (TEAC), nitric oxide (NO), and catalase (CAT) was measured and histopathological examination was conducted. Results in rat livers showed that TEAC level was decreased in rats receiving nanotubes at higher concentrations. Results in kidneys report that the level of NO showed higher concentration after long exposure, and results in animal serums showed lower levels of GSH in rats exposed to nanotubes at higher concentrations. The 18-month exposure also resulted in a statistically significant increase in GST activity in the group of rats exposed to nanotubes at higher concentrations compared to animals receiving MWCNTs at lower concentrations and compared to the control group. Therefore, an analysis of oxidative stress parameters can be a key indicator of the toxic potential of multi-walled carbon nanotubes.

Keywords: long-term toxicity; multi-walled carbon nanotubes; oxidative stress parameters; rats.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Examples of TEM images on multi-walled carbon nanotubes (MWCNT) used in our experiments. (a) MWCNT growth on a Si plate using Fe as a catalyst; (b) MWCNT in (spaghetti-like) growth process using Fe as a catalyst.
Figure 2
Figure 2
Schematic representation of the designed experiment.
Figure 3
Figure 3
(A) Fragment of liver with very well-preserved structure; (B) inset: closer view of the peripheral part of liver lobule with delicate (slight) features of small droplets steatosis. Primary objective magnification: A, 4×, B, 10×.; (C) Fragment of kidney with very well-preserved structure. There are some tubules with widened lumen; (D) inset: closer view of glomerulus without any significant changes. Primary objective magnification: A, 4×, B, 10×.
Figure 4
Figure 4
Comparison of total protein (TP) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months. (a)—statistically significant difference for the group receiving MWCNTs in concentration of 0.5 mg/mL versus 0.25 mg/mL in the livers (p = 0.022493); (b)—statistically significant difference for the group receiving MWCNTs in concentration of 0.5 mg/mL at 18 months versus 12 months in the livers (p = 0.032701); (c)—statistically significant difference for control group at 18 months versus control at 12 months in the livers (p = 0.030844); (d)—statistically significant difference for group receiving MWCNTs in concentration of 0.5 mg/mL versus 0.25 mg/mL in the kidneys (p = 0.0197); (e)—statistically significant difference for control group at 18 months versus control at 12 months in the kidneys (p = 0.008621).
Figure 5
Figure 5
Comparison of reduced glutathione (GSH) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months. (a)—statistically significant difference for control group at 18 months versus control at 12 months in the kidneys (p = 0.035571).
Figure 6
Figure 6
Comparison of thiobarbituric acid reactive substances (TBARS) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months. (a)—statistically significant difference for control group at 18 months versus control at 12 months (p = 0.025661).
Figure 7
Figure 7
Comparison of Trolox equivalent antioxidant capacity (TEAC) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months. (a)—statistically significant difference for the group receiving MWCNTs in a concentration of 0.25 mg/mL versus 0.03 mg/mL in the livers (p = 0.024330); (b)—statistically significant difference for the group receiving MWCNTs in a concentration of 0.5 mg/mL versus 0.25 mg/mL in the livers (p = 0.001632); (c)—statistically significant difference for the group receiving MWCNTs in nanotube concentration of 0.5 mg/mL at 18 months versus 12 months in the livers (p = 0.000702); (d)—statistically significant difference for control group at 18 months versus control at 12 months in the livers (p = 0.037120).
Figure 8
Figure 8
Comparison of nitric oxide (NO) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months. (a)—statistically significant difference for the group receiving MWCNTs in a concentration of 0.03 mg/mL at 18 months versus 12 months in the livers (p = 0.051588); (b)—statistically significant difference for MWCNTs in a concentration of 0.5 mg/mL versus control group in the kidneys (p = 0.0049); (c)—statistically significant difference for the group receiving MWCNTs in a concentration of 0.25 mg/mL at 18 months versus 12 months in the kidneys (p = 0.008052); (d)—statistically significant difference for the control group at 18 months versus control at 12 months in the kidneys (p = 0.000269).
Figure 9
Figure 9
Comparison of glutathione S-transeferase (GST) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months. (a)—statistically significant difference for MWCNTs in concentration of 0.5 mg/mL versus 0.25 mg/mL in the kidneys (p = 0.0178), (b)—statistically significant difference for MWCNTs in concentration of 0.25 mg/mL at 18 months versus 12 months in the kidneys (p = 0.041126).
Figure 10
Figure 10
Comparison of catalase (CAT) concentrations in the liver and kidneys of animals exposed to multi-walled carbon nanotubes for 12 and 18 months.
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