CeO2NPs relieve radiofrequency radiation, improve testosterone synthesis, and clock gene expression in Leydig cells by enhancing antioxidation

Abstract

Introduction: The ratio of Ce³⁺/Ce⁴⁺ in their structure confers unique functions on cerium oxide nanoparticles (CeO₂NPs) containing rare earth elements in scavenging free radicals and protecting against oxidative damage. The potential of CeO₂NPs to protect testosterone synthesis in primary mouse Leydig cells during exposure to 1,800 MHz radiofrequency (RF) radiation was examined in vitro. Methods: Leydig cells were treated with different concentrations of CeO₂NPs to identify the optimum concentration for cell proliferation. The cells were pretreated with the optimum dose of CeO₂NPs for 24 hrs and then exposed to 1,800 MHz RF at a power density of 200.27 µW/cm² (specific absorption rate (SAR), 0.116 W/kg) for 1 hr, 2 hrs, or 4 hrs. The medium was used to measure the testosterone concentration. The cells were collected to determine the antioxidant indices (catalase [CAT], malondialdehyde [MDA], and total antioxidant capacity [T-AOC]), and the mRNA expression of the testosterone synthase genes (Star, Cyp11a1, and Hsd-3β) and clock genes (Clock, Bmal1, and Rorα). Results: Our preliminary result showed that 128 μg/mL CeO₂NPs was the optimum dose for cell proliferation. Cells exposed to RF alone showed reduced levels of testosterone, T-AOC, and CAT activities, increased MDA content, and the downregulated genes expression of Star, Cyp11a1, Hsd-3β, Clock, Bmal1, and Rorα. Pretreatment of the cells with 128 μg/mL CeO₂NPs for 24 hrs followed by RF exposure significantly increased testosterone synthesis, upregulated the expression of the testosterone synthase and clock genes, and increased the resistance to oxidative damage in Leydig cells compared with those in cells exposed to RF alone. Conclusion: Exposure to 1,800 MHz RF had adverse effects on testosterone synthesis, antioxidant levels, and clock gene expression in primary Leydig cells. Pretreatment with CeO₂NPs prevented the adverse effects on testosterone synthesis induced by RF exposure by regulating their antioxidant capacity and clock gene expression in vitro. Further studies of the mechanism underlying the protective function of CeO₂NPs against RF in the male reproductive system are required.

Keywords: CeO2NPs radiofrequency radiation; Leydig cell; antioxidant; clock genes; testosterone synthesis.

Figure 1

Characterization of test material CeO₂NPs. (A) Scanning electron micrographs of CeO₂; (B) transmission electron micrographs of CeO₂; (C) size distribution histogram of CeO₂NPs; (D) the results of XRD test. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; XRD, X-ray powder diffractometry.

Figure 2

Distribution of SAR of the culture medium inside a standard petri dish (35-mm) exposed to 1,800 MHz radiofrequency fields (power densities, 200.27 μW/cm²). Abbreviation: SAR, specific absorption rate.

Figure 3

Stain of HSD-3β on primary leydig cells (A, 20×; B, 40×). Abbreviation: HSD-3β, 3β-Hydroxysteroid dehydrogenase.

Figure 4

Effects of different dose CeO₂NPs on the proliferation rate of primary Leydig cells. Notes: Compared with the 0 dose level (Solvent), the difference was significant as, *P<0.05, **P<0.01. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 5

Testosterone concentration in Leydig cells medium treated by CeO₂NPs, RF and CeO₂NPs + RF. Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, # P<0.05. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 6

CAT activity in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF. Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, # P<0.05, ## P<0.01. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation; CAT, catalase.

Figure 8

T-AOC levels in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF. Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, ## P<0.01. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation; T-AOC, total antioxidation capacity.

Figure 7

MDA content in Leydig cells treated by CeO₂NPs, RF and CeO₂NPs + RF. Notes: Compared with the Solvent Control group, the difference was significant as, **P<0.01; compared with the RF group at same exposure time, the difference was significant as, ## P<0.01. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation; MDA, malondialdehyde.

Figure 9

The mRNA expression of testosterone synthase genes (Star, Cyp11a1, Hsd-3β) in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF. Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, # P<0.05, ## P<0.01. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 10

The clock genes (Clock, Bmal1, Rorα) mRNA expression from Real-time PCR in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF. Notes: Compared with the Solvent control group, the difference was significant as, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, ## P<0.01. Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.