Aflatoxin B1 disrupts testicular development via the cell cycle-related Ras/PI3K/Akt signaling in mice and pig.

Aflatoxins B1 (AFB1), a type I carcinogen widely present in the environment, not only poses a danger to animal husbandry, but also poses a potential threat to human reproductive health, but its mechanism is still unclear. To address this question, multi-omics were performed on porcine Sertoli cells and mice testis. The data suggest that AFB1 induced testicular damage manifested as decreased expression of GJA1, ZO1 and OCCLUDIN in mice (p < 0.01) and inhibition of porcine Sertoli cell proliferation. Transcriptomic analysis suggested changes in noncoding RNA expression profiles that affect the cell cycle-related Ras/PI3K/Akt signaling pathway after AFB1 exposure both in mice and pigs. Specifically, AFB1 caused abnormal cell cycle of testis with the characterization of decreased expressions of CCNA1, CCNB1 and CDK1 (p < 0.01). Flow cytometry revealed that the G2/M phase was significantly increased after AFB1 exposure. Meanwhile, AFB1 downregulated the expressions of Ras, PI3K and AKT both in porcine Sertoli cell (p < 0.01) and mice testis (p < 0.01). Metabolome analysis verified the alterations in the PI3K/Akt signaling pathway (p < 0.05). Moreover, the joint analysis of metabolome and microbiome found that the changes of metabolites were correlated with the expression of flora. In conclusion, we have demonstrated that AFB1 impairs testicular development via the cell cycle-related Ras/PI3K/Akt signaling.

H3K4me3 as a target of di(2-ethylhexyl) phthalate (DEHP) impairing primordial follicle assembly.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastics additive that growing evidence indicates as endocrine disruptor able to negatively affect various reproductive processes both in female and male animals, including humans. However, the precise molecular mechanism of such actions is not completely understood. In the present study, scRNA-seq was performed on the ovaries of offspring from mothers exposed to DEHP from 16.5 days post coitum to 3 days post-partum, when the primordial follicle (PF) stockpile is established. While the histological observations of the offspring ovaries from DEHP exposed mothers confirmed previous data about a distinct reduction of oocytes enclosed in PFs. Focusing on oocytes, scRNA-seq analyses showed that the genes that mostly changed by DEHP were enriched GO terms related to histone H3-K4 methylation. Moreover, we observed H3K4me3 level, an epigenetics modification of H3 that is crucial for chromatin transcription, decreased by 40.28% (P < 0.01) in DEHP-treated group compared with control. When the newborn ovaries were cultured in vitro, the DEHP effects were abolished by tamoxifen (an estrogen receptor antagonist) or overexpression of Smyd3 (one specific methyltransferase of H3K4me3), in particular, the percentage of oocyte enclosed in PF was increased by 15.39% in DEHP plus Smyd3 overexpression group than of DEHP group (P < 0.01), which was accompanied by the upregulation of H3K4me3. Collectively, the present results discover Smyd3-H3K4me3 as a novel target of the deleterious ER-mediated effect of DEHP on PF formation during early folliculogenesis in the mouse and highlight epigenetics changes as prominent targets of endocrine disruptors like DEHP.

Oxidative stress as a plausible mechanism for zearalenone to induce genome toxicity.

Zearalenone (ZEN), a common non-steroidal estrogenic mycotoxin of the Fusarium genus, is one of the most frequent and powerful contaminant of grains and cereal products representing a serious threat for people and livestock health. In fact, ZEN causes cytotoxicity and genotoxicity in a variety of cell types at least in part through binding to estrogen receptors (ERs). The main pathways through which ZEN induces such effects remain, however, elusive. In particular, how the mycotoxin causes DNA damage, dysregulates DNA repair mechanisms, changes epigenome of targeted cells and, not least, affects chromatin conformation and non-coding RNA (ncRNA), is unclear. In the present paper, following extensive review of the literature about such ZEN effects and our own experience in studying the effects of this compound on reproductive processes, we propose that increased production of reactive oxygen species (ROS) and consequently oxidative stress (OS) are central in ZEN genotoxicity. Besides to shed light on the action mechanisms of the mycotoxin, this notion might help to develop effective strategies to counteract its deleterious biological effects.

Maternal Zearalenone exposure impacted ovarian follicle formation and development of suckled offspring.

Zearalenone (ZEN) is a secondary metabolite, which is mainly produced by Fusarium fungi and exists in various feeds and agricultural products. Recently, an increasing amount of data has shown that ZEN, as an estrogen-like hormone, can have harmful effects on the female reproductive system, especially on oogenesis and folliculogenesis. Breast milk is considered to be the ideal form of nutrition for infants; however, there are some records of contaminants in food, such as mycotoxins, which may be transferred from maternal blood to milk. In this study, we investigated the toxic effects of breast milk on folliculogenesis in offspring following maternal ZEN exposure. Our results showed that maternal ZEN exposure significantly inhibited the process of primordial follicle (PF) assembly and reduced the number of PFs in suckled offspring's ovaries. In addition, RNA-seq analysis showed that RIG-I-like receptor (RLRs) signaling pathways were activated after exposed to ZEN, which increased the expression levels of DNA damage (γ-H2AX, RAD51, and PARP1) and apoptosis related protein (BAX/BCL2 and Caspase-3). Finally, ZEN exposure interfered with follicular development, as evidenced by the reduced percentages of oocyte maturation and embryonic development when the offspring grew to adolescence. It is worth noting that maternal ZEN exposure disrupted the tri-methylation levels of H3K4, H3K9, and H3K27 in the offspring's oocytes. Our results indicated that maternal ZEN exposure affected ovarian development in offspring through the breast milk, which may be detrimental to their reproductive capability in adult life.

Inflammatory cytokines as key players of apoptosis induced by environmental estrogens in the ovary.

Natural and synthetic environmental estrogens (EEs), interfering with the physiological functions of the body's estrogens, are widespread and are rising much concern for their possible deleterious effects on human and animal health, in particular on reproduction. In fact, increasing evidence indicate that EEs can be responsible for a variety of disfunctions of the reproductive system especially in females such as premature ovarian insufficiency (POI). Because of their great structural diversity, the modes of action of EEs are controversial. One important way through which EEs exert their effects on reproduction is the induction of apoptosis in the ovary. In general, EEs can exert pro-and anti-apoptotic effects by agonizing or antagonizing numerous estrogen-dependent signaling pathways. In the present work, results concerning apoptotic pathways and diseases induced by representative EEs (such as zearalenone, bisphenol A and di-2-ethylhexyl phthalate), in ovaries throughout development are presented into an integrated network. By reviewing and elaborating these studies, we propose inflammatory factors, centered on the production of tumor necrosis factor (TNF), as a major cause of the induction of apoptosis by EEs in the mammalian ovary. As a consequence, potential strategies to prevent such EE effect are suggested.

Biotransformation products of phellopterin by rat liver microsomes and the inhibition on NO production in LPS-activated RAW264.7 cells.

Four new coumarins (2',3'-dihydroxyphellopterin, E-5-methoxytrichoclin acetate, Z-5-methoxytrichoclin acetate, and E-5-methoxytrichoclin) and three known coumarins (byakangelicol, byakangelicin, and Z-5-methoxytrichoclin) were produced by liver microsomes from rats pre-treated with sodium phenobarbital. The chemical structures were elucidated on the basis of their spectroscopic data. The inhibitory activities of nitric oxide (NO) production in lipopolysaccharide-activated macrophage-like cell line RAW264.7 were tested. The main biotransformation product, byakangelicin, showed inhibitory activities of NO production with the IC50 value of 217.83 µM, whereas the parent compound phellopterin showed cytotoxic effect on RAW264.7 cell at the concentration from 40 to 400 µM.