P–033 In vitro protective effect of α -tocopherol and anthocyanin against TiO2-NPs induced genotoxicity on human spermatozoa
Authors: Santonastaso M , Mottola F , Iovine C , Colacurci N , Rocco L 
Published: August 2021

Abstract

Study question
Do α -tocopherol and anthocyanin counteract human sperm DNA damage provoked by titanium dioxide nanoparticles (TiO2-NPs)? Summary answer: ↑-tocopherol and anthocyanin are able to counteract TiO2-NPs genotoxicity on human sperm cells reducing oxidative stress.

What is known already
The environmental release and the extensive use of TiO2-NPs have been implicated in poor human sperm functionality.TiO2-NPs is genotoxic on human sperm cells causing a loss of sperm DNA integrity, an increase of apoptotic process and a reduction of genomic stability related to an over production of intracellular ROS.Antioxidants are the substances that can scavenge free radicals. α -tocopherol, present in vegetables, is the most important lipophilic antioxidant involved in restore sperm parameters in several experimental models. Anthocyanin, present in Aronia melanocarpaand belonging to the flavonoid family, is able to prevent damage caused by varicocele-induced ROS in rats.

Study design, size, duration
Semen samples from 132 men were obtained by masturbation following 3–5 days sexual abstinence and were examined for sperm concentration, viability, motility and morphology according to WHO 2010. The sperm cells, after purification with 45–90% double density gradient, were exposed in vitro to 1 µg/L of TiO2-NPs, 1 µg/L of TiO2-NPs whit 1 mg/L of anthocyanin and 1 µg/L of TiO2-NPs plus 1 mg/L of α -tocopherolfor 15,30,45 and 90 minutes at 37 °C.

Participants/materials, setting, methods
Sperm motility and concentration were analyzed with Makler camber while sperm viability and morphology were evaluated by Eosin-Nigrosin Test and by Testsimplets® prestained slides respectively. Antigenotoxicity was evaluated by Comet assay, TUNEL test and RAPD-PCR technique and Genomic Template Stability (GTS,%) calculation. The intracellular ROS level was assessed by DFC Assay. The data were analyzed using ANOVA test by GraphPad Prism 6 and considered significant if p-value ≤ 0.05.

Main results and the role of chance
Sperm analyses showed none statistically significant changes in sperm viability and motility (progressive and non-progressive) for each treatment. Anthocyanin and α -tocopherol counteracted sperm DNA damage induced in vitro by TiO2-NPs neutralizing ROS in a time-dependent way. Comet assay displayed that both antioxidants reduced sperm DNA strand breaks produced by TiO2-NPs, in particular the damage was no longer statistically significant starting from 30 and 90 minutes of anthocyanin-TiO2-NPs and α-tocopherol-TiO2-NPs co-exposure respectively. The antioxidant supplementation induced a statistically decrease of sperm DNA fragmentation provoked by TiO2-NPs after 45 co-treatment minutes.The RAPD-PCR technique evidenced variations of bands number in the TiO2-NPs treated sperm compared to the negative control and anthocyanin and α -tocopherol-TiO2-NPs co-treated samples. Human sperm genomic stability increased after anthocyanin and α -tocopherol TiO2-NPs co-exposure respect to the TiO2-NPs single treatment, until it almost reaches the negative control at 90 minutes. Intracellular ROS percentage was significantly lower both in anthocyanin and α -tocopherol TiO2-NPs co-treated compared to TiO2-NPs alone starting from 45 minutes.

Limitations, reasons for caution
In vitro study. Wider implications of the findings: Our results showed a protective effect of anthocyanin and α -tocopherol on human DNA by neutralizing intracellular ROS induced by TiO2NPs. We suggest anthocyanin and α -tocopherol as suitable molecules to defend human sperm DNA from oxidative stress, with a potentially role in treatmentof male infertility due to environmental factors.

Trial registration number
None