Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles

Abstract

Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2'-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2'-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs.

Keywords: DNA methylation; PARP-1; oxidative stress; titanium dioxide nanoparticles.

Figure 1

Characterization of TNPs by TEM. Notes: Particle shape was analyzed by TEM (A) and the size distribution in the test media were evaluated by ImageJ software (B). Abbreviations: TNPs, titanium dioxide nanoparticles; TEM, transmission electron microscope.

Figure 2

Cell viability of A549 cells was measured by MTT assay after 24 hours TNP exposure. Notes: Cell viability was significantly decreased in a dose-dependent manner after TNP treatment. *P<0.05, compared with control group, n=5. Abbreviations: MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; TNP, titanium dioxide nanoparticle.

Figure 3

LDH activity was detected in TNP-treated A549 cells. Notes: After cells were exposed to TNPs (0 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL) for 24 hours, the supernatants were determined using a commercial LDH kit according to the manufacturer’s protocols. *P<0.05, compared with control group, n=5. Abbreviations: LDH, lactate dehydrogenase; TNPs, titanium dioxide nanoparticles.

Figure 4

Effects of TNPs on intracellular ROS generation in A549 cells. Notes: The typical picture of ROS detected by flow cytometry (A); the intracellular ROS level significantly increased in a dose-dependent manner (B). Data are expressed as mean ± SD. n=5, *P<0.05, compared with control. Abbreviations: FITC, fluorescein isothiocyanate; TNPs, titanium dioxide nanoparticles; ROS, reactive oxygen species; SD, standard deviation.

Figure 5

Effects of inhibitors, 5-aza or α-LA, on TNP-induced ROS generation in A549 cells. Notes: The methyltransferase inhibitor, 5-aza decreased TNPs-induced ROS generation (A); the antioxidant agent, α-LA suppressed ROS generation triggered by TNPs (B). Data are expressed as mean ± SD; n=5, *P<0.05, compared with control; ^#P<0.05, compared with TNPs. Abbreviations: 5-aza, 5-aza-2′-deoxycytidine; α-LA, α-lipoic acid; TNPs, titanium dioxide nanoparticles; ROS, reactive oxygen species; SD, standard deviation.

Figure 6

TNPs induced the alternation of PARP-1 methylation status. Notes: A549 cells were exposed to various concentrations (6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL) of TNPs for 24 hours, methylation-specific PCR was performed to examine the methylation status of PARP-1. U and M: primer sets specific to unmethylated (U) and methylated (M) DNA molecules. The data were typical examples of five independent experiments. *P<0.05, compared with control. Abbreviations: TNPs, titanium dioxide nanoparticles; PARP-1, poly(ADP-ribose) polymerase 1; PCR, polymerase chain reaction.

Figure 7

Effects of inhibitors, 5-aza or α-LA, on TNP-induced methylation of PARP-1 by MSP in A549 cells. Notes: Treatment with methyltransferase inhibitor, 5-aza (A); treatment with antioxidant agent, α-LA (B). U and M: primer sets specific to unmethylated (U) and methylated (M) DNA molecules. The data were typical examples of five independent experiments. *P<0.05, compared with control; ^#P<0.05, compared with TNPs. Abbreviations: 5-aza, 5-aza-2′-deoxycytidine; α-LA, α-lipoic acid; TNPs, titanium dioxide nanoparticles; PARP-1, poly(ADP-ribose) polymerase 1; MSP, methylation-specific PCR; PCR, polymerase chain reaction.