Effects Of Graphene Oxide Quantum Dots Exposure On GC-2/TM4 Autophagy And Metabolism In Male Germ Cells

Graphene oxide quantum dots(GOQDs)are kinds of zero-dimensional nanomaterials and have structures similar to graphene,but their lateral diameters are below 100nm.Compared with graphene,GOQDs show stronger quantum confinement effects and boundary effects which give them more unique features,such as better surface grafting,tunable luminescence emission and stable photolumine scence.Therefore,GOQDs are one of the most promising biomaterials in many fields,including bioimaging,drug delivery,DNA cutting systems,electrochemical biosensor and catalysis.Due to wide applications,the safety of the GOQDs usage is a natural concern.To date,some studies suggested that GOQDs had low toxicities in vitro and in vivo.Other studies discovered that GOQDs induced the release of reactive oxygen species,cell apoptosis,autophagy and DNA damage.Because of their tiny size and cell inflammatory response to them,nanoparticles can penetrate the blood-testis barrier easily and cause damages in male reproductive system.Besides,many studies showed that nanoparticles could disrupt cell autophagy which might play an important role in the survival of spermatozoa and was closely related to cell metabolism.However,studies on effects of GOQDs in male reproductive system are still lacking.GC-2 cell line and TM-4 cell line are originally derived from immortalized mouse spermatogonia and mouse Sertoli cells,respectively.Therefore,these two cell lines provide useful models for testing the chemical-induced male reproductive toxicities and the underlying mechanism in vitro.In this study,we studied the effects of GOQDs in male reproduction using GC-2 and TM4 cells and explored the molecular mechanisms from the perspective of cell autophagy and cell metabolism.Part ? Effects of Graphene oxide quantum dots on autophagy of GC-2 and TM4 cellsGraphene oxide quantum dots(GOQDs)have broad applications in many areas in cluding bioimaging,drug delivery,DNA cleavage system,sensors and photocatalyst.Recently increasing concerns have been raised about their biocompatibility,but studies on the effects of GOQDs on male germ cells are still lacking.In this work,we explored the effects and molecular mechanisms of GOQDs on two male germ cell lines(GC-2 and TM4).We found autophagosome accumulation in GC-2 and TM4 cells after GOQDs treatment.Both LC3-II/LC3-1 ratio and p62 levels increased,and the chloroquine-induced accumulation of LC3-II didn't enhance in the presence of GOQDs,which indicated that GOQDs blocked autophagic flux.Further studies found that the fusion between autophagosome and lysosome was not inhibited by GOQDs,but the proteolytic capacity of lysosome was weakened and both the expression and activity of cathepsin B reduced.Taken together,these results showed that GOQDs blocked autophagic flux by decreasing the amount and enzymatic activity of cathepsin B and inhibiting lysosome proteolytic capacity in GC-2 and TM4 cells,which might have a potential hazard to male reproduction.Part ? Effects of Graphene oxide quantum dots on metabolism of GC-2 and TM4 cellsMetabolomics,a high-throughput genomics analysis method,can be used to detect the changes of small metabolites and metabolic pathways under the state of health or disease,which contributed to predicting the toxicities of chemicals and clarifing the biological mechanisms of exogenous chemicals.In this study,we further explored the effects and molecular mechanisms of GOQDs on GC-2 and TM4 cells by metabolomics.The results showed that,after GOQDs exposure,a variety of small metabolites changed in both GC-2 and TM4 cells.Pathway enrichment analysis showed that protein synthesis pathway changed significantly.There were also many small metabolites changed in the pathways of oxidation of branched chain fatty acids,P-oxidation of very long chain fatty acids and purine metabolism.In addition,we found that intracellular ATP levels decreased significantly after GOQDs exposure.Many studies showed that autophagy was closely related to cell metabolism,so the change of protein synthesis pathway was probably due to the blockage of autophagic flux by GOQDs,which inhibited the degradation of protein aggregates,leading to intracellular amino acid disorder and affecting protein synthesis.The activation of oxidation of branched chain fatty acids pathway and ?-oxidation of very long chain fatty acids pathway may be feedback regulations owing to the decrease of cellular ArTP levels.Our study is the first to explore the effects and mechanisms of GOQDs on GC-2 and TM4 cells by metabolomics and provides a new theoretical foundation for further studies.