Hexafluoropropylene oxide (HFPO) homologues (HFPOs), specifically HFPO-dimeric acid (DA), HFPO-trimeric acid (TA) and HFPO-tetrameric acid (TeA), have emerged as industrial replacements for phased-out perfluorooctanoic acid (PFOA), garnering considerable attention due to their environmental ubiquity and bioaccumulation potential. Nevertheless, the reproductive toxicity of HFPOs remains incompletely characterized, particularly regarding their endocrine-disrupting effect and the underlying mechanisms involving Leydig cell dysfunction. In this study, we investigated the cytotoxic influences of HFPOs on TM3 Leydig cells, focusing on mitochondrial function and dynamics, oxidative stress, and apoptosis. Our findings demonstrated that exposure to HFPOs significantly compromised mitochondrial function and fusion-fission dynamics by disrupting the SIRT1/PGC1α signaling pathway. The mitochondrial dysfunction further triggered excessive ROS production and apoptosis, ultimately impairing TM3 Leydig cell viability and testosterone secretion. However, supplementation with the SIRT1 agonist SRT1720 relieved the inhibitory effect of HFPOs on SIRT1/PGC1α signaling pathway and reversed the expression of apoptosis-associated proteins (BAX/BCL2), oxidative stress-associated proteins (SOD1/SOD2), as well as proteins associated with mitochondrial fusion (MFN2/OPA1) and fission (DRP1/FIS1). These results elucidated the involvement of the SIRT1/PGC1α pathway in mediating the cytotoxicity of HFPOs. Notably, the activation of SIRT1 mitigated HFPO-induced toxicity in the TM3 cells, uncovering its potential in safeguarding testicular cells from the damage caused by HFPOs exposure.
Ethnopharmacological relevance Crassostrea gigas Thunberg and other oysters have been traditionally used in China as folk remedies to invigorate the kidney and as natural aphrodisiacs to combat male impotence. Aim of the study Erectile dysfunction (ED) has become a major health problem for the global ageing population. The aim of this study is therefore to evaluate the effect of peptide-rich preparations from C. gigas oysters on ED and related conditions as increasing evidence suggests that peptides are important bioactive components of marine remedies and seafood. Materials and methods Crassostrea oyster peptide (COP) preparations COP1, COP2 and COP3 were obtained from C. gigas oysters by trypsin, papain or sequential trypsin-papain digestion, respectively. The contents of testosterone, cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) and the activity of nitric oxide synthase (NOS) in mice and/or cells were measured by enzyme-linked immunosorbent assays. Real-time PCR was used to assess the expression of genes associated with sex hormone secretion pathways. The model animal Caenorhabditis elegans was also used to analyze the gene expression of a conserved steroidogenic enzyme. In silico analysis of constituent peptides was performed using bioinformatic tools based on public databases. Results The peptide-rich preparation COP3, in which >95% peptides were <3000 Da, was found to increase the contents of male mouse serum testosterone and cAMP, both of which are known to play important roles in erectile function, and to increase the activity of mouse penile NOS, which is closely associated with ED. Further investigation using mouse Leydig-derived TM3 cells demonstrates that COP3 was able to stimulate the production of testosterone as well as NO, a pivotal mediator of penile erection. Real-time PCR analysis reveals that COP3 up-regulated the expression of Areg and Acvr2b , the genes known to promote sex hormone secretion, but not Fst , a gene involved in suppressing follicle-stimulating hormone release. Furthermore, COP3 was also shown to up-regulate the expression of let-767 , a well-conserved C. elegans gene encoding a protein homologous to human 17-β-hydroxysteroid dehydrogenases. Preliminary bioinformatic analysis using the peptide sequences in COP3 cryptome identified 19 prospective motifs, each of which occurred in more than 10 peptides. Conclusions In this paper, Crassostrea oyster peptides were prepared by enzymatic hydrolysis and were found for the first time to increase ED-associated biochemical as well as molecular biology parameters. These results may help to explain the ethnopharmacological use of oysters and provide an important insight into the potentials of oyster peptides in overcoming ED-related health issues.
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