Titanium Dioxide Nanoparticles: Effects on Development and Male Reproductive System

Abstract

Titanium dioxide nanoparticles (TiO₂-NPs) are used intensively. Thanks to their extremely small size (1-100 nm), TiO₂-NPs are more absorbable by living organisms; consequently, they can cross the circulatory system and then be distributed in various organs including the reproductive organs. We have evaluated the possible toxic effect of TiO₂-NPs on embryonic development and the male reproductive system using Danio rerio as an organism model. TiO₂-NPs (P25, Degussa) were tested at concentrations of 1 mg/L, 2 mg/L, and 4 mg/L. TiO₂-NPs did not interfere with the embryonic development of Danio rerio, however, in the male gonads the TiO₂-NPs caused an alteration of the morphological/structural organization. The immunofluorescence investigation showed positivity for biomarkers of oxidative stress and sex hormone binding globulin (SHBG), both confirmed by the results of qRT-PCR. In addition, an increased expression of the gene responsible for the conversion of testosterone to dihydrotestosterone was found. Since Leydig cells are mainly involved in this activity, an increase in gene activity can be explained by the ability of TiO₂-NPs to act as endocrine disruptors, and, therefore, with androgenic activity.

Keywords: Danio rerio; TiO2-NPs; embryonic development; endocrine system; male infertility; testis.

Figure 1

SEM images of TiO₂-NP. Marker is 1 µm (a), and 200 nm (b). (c) Autocorrelation function of the colloidal solution as prepared solution and after 24 h of sedimentation.

Figure 2

(a) Zebrafish embryo unexposed to TiO₂-NPs (negative controls); (b) zebrafish embryo exposed to 3,4-dichloroaniline (positive controls). Zebrafish embryo exposed to 1 mg/L (c); 2mg/L (d); and 4 mg/L (e) TiO₂- NPs. Scale bar 410 µm.

Figure 3

Phenotypes of larvae exposed to TiO₂-NPs and the unexposed group from 24 to 96 hpf. Scale bar 420 µm.

Figure 4

In order (1) PARP-1 antibody-staining, (2) Hsp70 antibody-staining, and (3) MT antibody-staining. (a,e,i) larva unexposed to TiO₂-NPs; (b,f,l) larva exposed to 1 mg/L TiO₂-NPs; (c,g,m) larva exposed to 2 mg/L TiO₂-NPs; and (d,h,n) larva exposed to 4 mg/L TiO₂-NPs. The histogram next to each photo represents the average fluorescence intensity (AU) of the corresponding biomarker. Scale bar 420 µm.

Figure 5

In order (1) SHBG antibody-staining and (2) PTMA antibody-staining. (a,e) larva unexposed to TiO₂-NPs; (b,f) larva exposed to 1 mg/L TiO₂-NPs; (c,g) larva exposed to 2 mg/L TiO₂-NPs; and (d,h) larva exposed to 4 mg/L TiO₂-NPs. The histogram next to each photo represents the average fluorescence intensity (AU) of the corresponding biomarker. Scale bar 420 µm.

Figure 6

Histological section of gills. (a) CTRL gills. Exposed groups to (b) 2mg/L; and (c) 4mg/L TiO₂-NPs. Arrows indicate the secondary lamellae, that showed a hyperplasia in the exposed groups, while it is not appeared in the control groups. Scale bar 1500 µm.

Figure 7

Histological sections of the testis. Unexposed group: 10× (a), and 40× (b). Group of 2mg/L TiO₂-NPs: (c) 10×, and (d) 40×. E-E staining, sections 4 µm. Blue arrow indicate the detachment of the spermatogenic epithelium from the connective tissue in the exposed group (2mg/L), while in the control group the spermatogenic epithelium maintains its contact with connective tissue (green arrow). Scale bar 260 µm.

Figure 8

Histological sections of testis staining with toluidine blue (sections 0.85 µm). Unexposed group: 10× (a), and 40× (b). Group of 2mg/L TiO₂-NPs: (c) 10×, and (d) 40×. Red * indicates the area occupied by spermatozoa into the tubules. Scale bar 260 µm.

Figure 9

(a) (10×) and (b) (40×) unexposed group with good morphology and organization of tubules testis; (c) (10×) and (d) (40×) group exposed to 4mg/L TiO₂-NPs with disorganization of tubules testis. Green arrows indicate the intact and well organization of tubules testis, while the red arrows indicate their disordered organization. Scale bar 260 µm.

Figure 10

Ultrathin sections of zebrafish testis. (a) Section of testis unexposed; (b) section of testis exposed to 4 mg/L with evident vesiculation. Red * indicate the Sertoli cells that showed a evident vesiculation in the exposed group compared to control. Scale bar 2 µm.

Figure 11

SHBG expression in testis tissue. Nuclei blue (DAPI) and red fluorescent of SHBG protein. Scale bar 265 µm.

Figure 12

Results of qRT-PCR of all genes investigated.

Figure 13

P540 expression in testis tissue. Nuclei blue (DAPI) and red fluorescent of P540 protein. Scale bar 265 µm.

Figure 14

2D-depiction shows the interactions that characterize the pocket.