Document Type : Original Article
Authors
1 Department of Histology, Faculty of Medicine, Al-Azhar University, Assiut, Egypt;Department of Pathology, Insaniah University, Kuala Ketil Kedah, Darul Aman, Malaysia
2 Forensic Unit, Department of Pathology, National University of Malaysia Medical Centre, Jalan Yaakob Latif, Bandar Tun Razak, Kuala Lumpur, Malaysia;4Department of Forensic Medicine and Clinical Toxicology, Faculty of M
3 5Department of Anatomy, National University of Malaysia Medical Centre, Jalan Yaakob Latif, Bandar Tun Razak, Kuala Lumpur, Malaysia
Abstract
Keywords
Titanium dioxide (TiO2), also recognized as titanium
oxide or titania, is a naturally occurring oxide of titanium
which is commonly used as a colouring pigments to
provide a white colour in products such as dyes, plastic,
paper, and foods (
The lethal effects of nanoparticles can be accredited to
their small size and hence outsized surface area which
results in increased rates of chemical reaction and infiltration
into the cells interfering with numerous subcellular
physiological mechanisms (
Previous studies have established that TiO2.
cles may pass into the cells of the reproductive system
and induce damage (
N-acetylcysteine (NAC) is an antioxidant and free-radical
scrounger. It acts as a cysteine contributor and upholds
or even upsurges the intracellular levels of glutathione
(GSH) (a tripeptide which guards cells against toxins).
NAC is characterized by its antioxidant ability through
releasing sulfhydryl groups which in turn, reduce ROS
levels and possess the ability to reduce oxidative stress
directly, suppress the nuclear factor kappa b (NF-.B) inflammation
pathway and inflammatory cytokines secretion,
and enhance the GSH production (
The current experimental study was carried out in Animal Laboratory, Zagazig University. TiO2 nanoparticles (Titanos, China) were nanopowder of 21 nm size with =99.5% purity dissolved in gum acacia solution. NAC was purchased from SEDICO, Egypt. Fifty male albino rats weighing 150-200 g were obtained from the Animal House, Faculty of Medicine, Zagazig University. The study was conducted in accordance with the guidelines of the Ethics Committee for Research of Zagazig University. The rats were divided into 5 groups. Group I was the control group that received no treatment. Group II was treated with 1 ml of 5% gum acacia solution (the solvent used for titanium dioxide) by oral gavage once daily for 12 weeks. Group III was orally treated with 100 mg/kg of NAC once daily for 12 weeks. Group IV was orally treated with 1200 mg/ kg of TiO2 nanoparticles once daily for 12 weeks. Group V was orally treated with a combination of 100 mg/kg of NAC and 1200 mg/kg of TiO2 nanoparticles once daily for 12 weeks.
For histopathological analysis, the left testis was fixed
in Bouin’s solution and the tissue was processed and embedded
in paraffin blocks for preparation of 5-µm thick
sections. Sections were stained with Haematoxylin and
Eosin and examined by light microscopy. Ultrastructural
examination of the left testis was conducted using the
Transmission Electron Microscope (TEM). The analysis
was performed according to the method described by
Glauret and Lewis (
Blood was collected from the tail vein then it was centrifuged
to collect serum. Total testosterone level was
measured by enzyme-linked immunosorbent assay (ELISA).
Malondialdehyde (MDA) was estimated by the thiobarbituric
acid assay. Estimation of reduced GSH level
was done using 5, 5’-dithiobis nitro benzoic acid assay.
The comet assay was performed according to the method
of Singh et al. (
Data were analyzed using Statistical Package for the Social Sciences software (SPSS version 22.0, IBM, USA). Differences between multiple means (quantitative variables) were evaluated by one way ANOVA test, followed by LSD. A P<0.05 was considered statistically significant.
Macroscopic examinations of the left testis in terms of
color, testis to body weight ratio, and infarction of treated
groups revealed no significant changes compared to the control
group. Histopathological examination of groups I, II,
and III showed the same histological features without any
abnormal histopathological finding such as dark nuclei, hyaline
fluids, and blood extravasation into the interstitial spaces
(
Photomicrograph showing histology of seminiferous tubules. A. Group I, II, and B. Group III showing normal seminiferous tubules lined by spermatogonia (Sg) close to the basal membrane (arrow), spermatogenic cells (Sc) with many mitotic figures (M) and Sertoli cells (arrow head). Seminiferous tubules lumen containing spermatid (Sp) with normal interstitial tissue (Ic) in between, C. Group IV showing marked disorganization, spermatogenic cells with dark pyknotic nuclei (tailed arrow), interstitial cells (Ic), basement membrane separation in many areas (arrow), extensive area between seminiferous tubules, hyaline exudate (H), and extravasation of blood (RBCs) in the interstitium, and D. Group V showing: separation of basement membrane of seminiferous tubules (arrow) and hyaline exudate (H) in the interstitium (H&E: ×200).
Photomicrograph showing electron microscopy of a seminiferous tubule. A, B. Group I, II ×4000, ×8000, C, and D. group III ×4000, showing spermatogenic cells (Sc) with its euchromatic nucleus (N). The cytoplasm contains mitochondria (M) and lysosomes (Ly); spermatogonia (Sg) resting on the basement membrane (Bm) with adjacent blood capillary (Bc) and interstitial cell (ISC); and a spermatid (Sr) with its acrosomal cap (A) and numerous vesicles (Vs).
Photomicrograph showing electron microscopy of a seminiferous tubule. A, B. Group IV ×4000 showing irregular and thickened basement membrane (Bm), multi-folded euchromatic nuclei (N) with marked indentation (waved arrow), marked cytoplasmic vacuolation, mitochondria (M), lysosomes (Ly) and blood capillary (Bc), C, and D. Group V ×4000 showing vacuolation (V), spermatogonia (Sg) early spermatids (Sr) with their euchromatic nuclei and numerous vesicles (Vs), flattened myoid cell (My) and collagen fibers deposition (square) in the basement membrane (Bm).
Using TEM, groups I, II, and II showed normal ultrastructures
including normal seminiferous tubules lined
with spermatogonia close to the basal membrane, spermatogenic
cells with many mitotic figures and sertoli
cells (
Photomicrograph showing immunohistochemistry of seminiferous tubules. A. Group I, II, B. group III showing weak TNF-a immuno-reactivity, C. Group IV showing strong positive TNF-a immunoreaction (arrow), and D. Group V showing decreased TNF-a immuno-reactivity (×200).
There was an insignificant difference in total testosterone
level among groups I, II, and III. However, the total
testosterone level of group IV displayed a significant decrease
compared to groups I, II, and III. Total testosterone
level was significantly decreased in group V compared to
groups I, II, and III . Meanwhile, there was a significant
increase in total testosterone level of group V compared
to group IV (
Total testosterone, serum reduced glutathione (GSH), and serum malondialdehyde (MDA)
Biochemical parameters | Group I | Group II | group III | Group IV | Group V |
---|---|---|---|---|---|
Total testosterone (nmol/L) | 23.8 ± 8.8bc | 21.2 ± 8.6bc | 26.2 ± 8.8bc | 0.38 ± 0.0ac | 15.4 ± 6.0ab |
GSH (nmol/L) | 50.2 ± 4.0bc | 49.1 ± 3.9bc | 52.8 ± 4.7bc | 31.8 ± 5.7ac | 61.9 ± 3.5ab |
MDA (nmol/L) | 74.9 ± 3.5bc | 75.0 ± 3.9bc | 73.9 ± 3.4bc | 136.3 ± 21ac | 85.7 ± 6.6ab |
Values are referred as mean ± SD. P<0.05 was considered statistically significant.
a; significance with group I ,II and III, b; significance with group IV, and c; Significance with the group V.
Photomicrograph of comet test showing nuclei of testes cells. A. Group I, II, B. group III showing almost normal condensed type nuclei and undamaged cells, C. Group IV showing abnormal tailed nuclei and damaged cells (white arrow), and D. Group V showing less number of abnormal tailed nuclei and damaged cells.
According to comet assay results, cells nuclei of group IV
showed a significant increase in percentage (%) of tailed nuclei,
tail length, tail DNA% and unit tail moment compared
to groups I, II, and III. Co-administration of NAC and TiO2
nanoparticles caused a significant decrease in DNA damage
parameters in group V compared to group IV. However,
there was a significant increase (P<0.05) in % of tailed nuclei,
tail length, tail DNA % and unit tail moment of group V
compared to groups I, II, and III (
Prevalent applications of nanomaterial cause a huge
potential for human exposure to these compounds. However,
many experts and establishments have upstretched
the environmental and toxicological concerns regarding
nanotechnology (
Our results are supported by data reported by Takeda et
al. (
In the present study, examination of the testis sections
of TiO2 nanoparticles-treated group revealed signs of inflammatory
damage in the testicular tissue. For instance,
administration of NAC along with TiO2 nanoparticles
showed partial improvement in testicular tissue, which was
found by histological and immunohistochemical examination.
However, cytoplasmic vacuolation was still observed
with mild collagen fibers deposition in the basement membrane.
From an ultrastructural point of view, variable sized
intercellular spaces were observed. El Ghazzawy et al. (
Co-administration of NAC and TiO2 nanoparticles led to
a reduction in TNF-a immunoreactivity. Our results are in
accordance with those indicated by Park et al. (
Co-administration of NAC and TiO2 resulted in a significant
increase in GSH. These results showed a time-dependent
reduction in GSH level in TiO2 nanoparticles-treated
rats. Similar findings were reported by Long et al. (
There was a significant rise in MDA level in TiO2 nanoparticles-
treated group compared to the group treated
with NAC+TiO2. However, MDA levels in NAC+TiO2
nanoparticles-treated group were significantly higher than
those of the control group. Significant changes in MDA
levels suggest that induction of pathological lesions is
probably mediated through the oxidative stress enhanced
by the dumped nanoparticles. These results were consistent
with those reported by Attia et al. that showed a time-
dependent significant release of oxidative stress in the liver
as evident by increased MDA and reduced GSH levels
(
The comet assay is a broadly used assay for investigation
of DNA damage and repair, genotoxic properties of
chemicals and pharmaceuticals, environmental biomonitoring,
and also human monitoring. However, comet assay
has been used for determination of the toxicity of
highly reactive nanoparticles and several studies used it
to test the potential toxicity of manufactured nanoparticles
by assessing DNA strand breaks or oxidative DNA
lesions (
Furthermore, TiO2 nanoparticles in aqueous suspension
release free radicals which can result in DNA damage
by oxidation, nitration, methylation or deamination
reactions (
NAC acts as an antioxidant through expanding the synthesis
of endogenous GSH which is frequently exhausted
as a result of augmented oxidative stress (
Oral administration of TiO2 nanoparticles induced toxic effects and DNA damage in the testes and these adverse effects may be attributed to induction of oxidative stress. Administration of NAC along with TiO2 nanoparticles, protected against TiO2 damaging effect.