P-266 Profiling redox effects induced by micro-nanoplastics in the female reproductive system: insights from human granulosa cells
Authors: Bianchi A , Casoli G , Vergara T , Cordone V , Placidi M , Tatone C , Di Emidio G 
Published: July 2024

Abstract

Study question
Are nano-microplastics (NMPs) taken up into human granulosa cells causing redox alterations?

Summary answer
NMPs enter into human granulosa cells and influence viability, energy production and antioxidant response.

What is known already
In recent decades, anthropogenic activities have increased the production of global plastic with millions of tons produced every year. Under the action of different physical, chemical or biological agents, plastic waste breaks down into (NMP) particles that propagate in the environment posing threats for human health. NMPs translocate from digestive tract to circulatory systems reaching organs and cells, including female gonads. Nevertheless, there is insufficient knowledge of their effects on reproductive functions in mammals to allow for an accurate risk assessment to be conducted and any risks managed in animal and human being contexts.

Study design, size, duration
Human ovarian granulosa cells (KGN cell line) were exposed to NMPs of different sizes (40 nm, 70 nm, 100 nm, and 200 nm) at concentrations ranging from 5 to 1000 μg/ml for 24 h and then processed for cellular and biochemical analyses

Participants/materials, setting, methods
NMPs uptake by granulosa cells was tested by the employment of fluorescent NMPs and observation under confocal laser scanning microscopy. Cell viability was assed by Cell Counting Kit8. The effects of NMPs on mitochondrial bioenergetics was evaluated by MitoStress kit (Seahorse Xfe96, Agilent). ATP production was evaluated by Cell Titer-Glo ATP assay kit (Promega). Key enzymes of antioxidant response were analysed at transcript and protein level by using real-time Taqman PCR and Western blotting, respectively.

Main results and the role of chance
We demonstrated that NMPs are taken up by granulosa cells at all tested concentrions along with significant decrease of cell vitality with all sizes and concentrations. Seahorse analysis revealed an altered bioenergetic profiles. ATP levels increase at 70, 100 and 200 nm (one way ANOVA p = 0.001) at all tested concentrations (one way ANOVA p &lt; 0.001).
The gene and protein expression of catalase (CAT) superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) increased at 5 and 100 µg/ml with all sizes except 100 nm, whereas the level of sirtuins (SIRT1 and SIRT3) transcripts and protein decreased. Reduced levels of phosphorylated NRF2, the transcription factor that activates antioxidant responsive elements (ARE)-mediated gene expression were observed.

Limitations, reasons for caution
The use of human cell lines requires careful consideration when transferring to human testing.

Wider implications of the findings
Our results contribute to understanding the effects of NMPs on mammalian fertility in order to find possible protective approaches. Considering that the level of susceptibility of mammalian female germ cells to NMPs is still unknown, present results contribute to evidence-based strategies allowing living and working in a health promoting environment.

Trial registration number
Effects of combined exposure to nano/microplastics and plastic additives on mammalian female fertility (CUP E53D2301100006)