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.

Single-cell transcriptomic profiling to evaluate the effects of Di(2-ethylhexyl)phthalate exposure on early meiosis of female mouse germ cells.

Di(2-ethylhexyl)phthalate (DEHP) has proven characteristics of an endocrine-disrupting compound (EDC), which can threaten the reproductive health of humans and other animals. In mammals, a series of chromosomal events occur during the meiotic stage of oocytes. External toxins may enter the body and cause infertility and other related diseases. Therefore, it is crucial to explore the influence of DEHP exposure on the molecular mechanism of germ cell meiosis. We used single-cell RNA sequencing (scRNA-seq) to analyse the ovaries of foetal mice at embryonic day 12.5 (E12.5) and E14.5 after maternal DEHP exposure. DEHP exposure further activated the pathways related to DNA repair in germ cells, increased the expression of genes related to DNA damage and changed the developmental trajectory of germ cells. DEHP exposure may affect the proliferation of pregranulosa (PG) cells. Moreover, DEHP exposure altered the signal transduction between PG cells and germ cells. We showed that DEHP affects meiosis by causing DNA damage in oocytes and disrupting the signal transduction between PG cells and germ cells. These results provide a strong theoretical basis for the prevention and treatment of DEHP-mediated female reproductive health problems.

Identification of apoptotic pathways in zearalenone-treated mouse sertoli cells.

Zearalenone (ZEN), one of the most prevalent non-steroidal oestrogenic mycotoxins, is primarily produced by Fusarium fungi. Due to its toxicity as an oestrogenic compound and wide distribution in feed and foods, the reproductive toxicology of ZEN exposure is of public concern. The aim of the present study was to investigate the effect of ZEN on Sertoli cells to identify apoptotic pathways induced by this compound. We found that ZEN reduced the viability and caused apoptosis in Sertoli cells in vitro. Notably, we observed that such effects were associated with a significant increase in reactive oxygen species (ROS) and the number of cells that showed positive staining for γH2AX and RAD51, enzymes essential for repairing DNA damage. There was a parallel decrease in the expression of occludin and connexin 43, proteins that are present in the testis-blood barrier and gap junctions of Sertoli cells, respectively. Overall, the present study confirms that ZEN exposure can have serious deleterious effects on mammalian Sertoli cells and offers novel insight about its molecular targets in these cells.

Impaired primordial follicle assembly in offspring ovaries from zearalenone-exposed mothers involves reduced mitochondrial activity and altered epigenetics in oocytes.

Previous works have shown that zearalenone (ZEA), as an estrogenic pollutant, has adverse effects on mammalian folliculogenesis. In the present study, we found that prolonged exposure of female mice to ZEA around the end of pregnancy caused severe impairment of primordial follicle formation in the ovaries of newborn mice and altered the expression of many genes in oocytes as revealed by single-cell RNA sequencing (scRNA-seq). These changes were associated with morphological and molecular alterations of mitochondria, increased autophagic markers in oocytes, and epigenetic changes in the ovaries of newborn mice from ZEA-exposed mothers. The latter increased expression of HDAC2 deacetylases was leading to decreased levels of H3K9ac and H4K12ac. Most of these modifications were relieved when the expression of  Hdac2 in newborn ovaries was reduced by RNA interference during in vitro culture in the presence of ZEA. Such changes were also alleviated in offspring ovaries from mothers treated with both ZEA and the coenzyme Q10 (CoQ10), which is known to be able to restore mitochondrial activities. We concluded that impaired mitochondrial activities in oocytes caused by ZEA are at the origin of metabolic alterations that modify the expression of genes controlling autophagy and primordial follicle assembly through changes in epigenetic histones.

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.

Single-cell transcriptomic profiling provides insights into the toxic effects of Zearalenone exposure on primordial follicle assembly.

Rationale: Zearalenone (ZEN), a pollutant in our daily diet, seriously threatens the reproductive health of humans and animals. The primordial follicle (PF) assembly in the mouse occurs during the perinatal period, which determines the whole ovarian reserve in reproductive lifespan. In the current investigation, we administered ZEN orally to perinatal mice from 16.5 days post coitum (dpc) to postnatal day 3 (PD3), and single-cell RNA sequencing (scRNA-seq) was performed on PD0 and PD3 ovarian tissues in the offspring to check ZEN toxic to primordial follicle formation at the single cell level. Methods: Ovarian tissues (in vivo) were examined by single cell RNA sequencing analysis, Immunostaining, and Western blotting. Ovarian tissues (in vitro) were examined by qRT-PCR, Immunostaining, and Western blotting. Results: We found that ZEN exposure altered the developmental trajectory of both germ cells and granulosa cells. Furthermore, after establishing the cell-cell communication network between germ cells and granulosa cells, we found that this was perturbed by ZEN exposure, especially during the Hippo signaling pathway. Conclusions: This study showed that ZEN affected the status of germ cells and granulosa cells through the Hippo signaling pathway and blocked the assembly of PFs. This research contributes to our deeper understanding of the mechanisms of toxicity in different cell types and the disruption of normal intercellular signaling by ZEN exposure.

Two new phenolic glycosides from the stem of Zanthoxylum armatum DC.

Two new phenolic glycoside, 2-methoxy-4-hydroxylphenyl-1-O-α-L-rhamnopyranosyl- (1″ â†’ 6')-ß-D-glucopyranoside. (1) and threo-3-methoxy-5-hydroxy-phenylpropanetriol-8-O-ß-D-glucopyranoside (2), were isolated from the stems of Zanthoxylum armatum. The compounds 1 and 2 showed weak scavenging activity in DPPH free radical assay with IC50 values of 323 and 114 mM, respectively.

Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats.

BACKGROUND: The roots of Angelica dahurica cv. Qibaizhi is frequently used in clinical practice as a traditional Chinese medicine. However, a comprehensive study of the pharmacokinetics of this medicine has not been carried out. METHOD: A sensitive and specific liquid chromatographic-tandem mass (LC-MS/MS) spectrometric method was established to investigate pharmacokinetics of sixteen coumarins of Angelicae dahuricae Radix (ADR) in rat plasma, including xanthotoxol (1), oxypeucedanin hydrate (2), 5-hydroxy-8-methoxypsoralen (3), (-)-marmesin (4), byakangelicin (5), columbianetin (6), psoralen (7), xanthotoxin (8), neobyakangelicol (9), isoimpinellin (10), bergapten (11), heraclenin (12), oxypeucedanin ethanolate (13), imperatorin (14), phellopterin (15), isoimperatorin (16). Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction-mode (MRM). RESULTS: The method established in this assay was successfully applied to the pharmacokinetic study of the selected coumarins in rat plasma after oral administration of the extract of ADR, and the pharmacokinetic characteristics of sixteen coumarins were clearly elucidated. CONCLUSION: This pharmacokinetic identification of multiple coumarins of ADR in rats provides a significant basis for better understanding the metabolic mechanism of the herb medicine.

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.

[Study on the determination of Cd and Pb in Radix Salviae Miltiorrhizae by ICP-AES].

In the present paper, a method was developed for the determination of Cd and Pb by ICP-AES. The effects of two different sample treatment methods, including wet digestion method and dry ash method, were compared. The contents of Cd and Pb in Danshen (Radix Salviae miltiorrhizae) grown in some areas of Shandong were determined. The results show that it is satisfactory to apply ICP-AES to the determination of Cd and Pb with the detection limits (DL) of 1.92 ng x mL(-1) and 1.07 ng x mL(-1), the precision values (RSD) of 3.14% and 1.83%, and the recovery rates of 103.0% and 96.2% respectively. The wet digestion method is better than the dry ash method for its lower RSD and higher recovery rate. The dry ash method is suitable for the determination of Pb, but not suitable for the determination of Cd due to its low recovery rate. The results also show that the contents of Cd and Pb in Danshen are lower than the standard of Chinese Pharmacopoeia (2005), which meets the standard of GAP production.