Effects of aflatoxin and fumonisin on gene expression of growth factors and inflammation-related genes in a human hepatocyte cell line.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are mycotoxins widely distributed in maize and maized-based products, often occurring together. The implications of co-exposure to aflatoxin and fumonsin for human health are numerous, but a particular concern is the potential of FB1 to modulate AFB1 hepatotoxicity. This study evaluated the toxicity of these mycotoxins, alone or combined, in a human non-tumorigenic liver cell line, HHL-16 cells, and assessed the effects of AFB1 and FB1 on expression of genes involved in immune and growth factor pathways. The results demonstrated that in HHL-16 cells, both AFB1 and FB1 had dose-dependent and time-dependent toxicity, and the combination of them showed a synergistic toxicity in the cells. Moreover, AFB1 caused upregulation of IL6, CCL20, and BMP2, and downregulation of NDP. In combination of AFB1 with FB1, gene expression levels of IL6 and BMP2 were significantly higher compared to individual FB1 treatment, and had a tendency to be higher than individual AFB1 treatment. This study shows that FB1 may increase the hepatoxicity of AFB1 through increasing the inflammatory response and disrupting cell growth pathways.

Trophectoderm non-coding RNAs reflect the higher metabolic and more invasive properties of young maternal age blastocysts.

Increasing female age is accompanied by a corresponding fall in her fertility. This decline is influenced by a variety of factors over an individual's life course including background genetics, local environment and diet. Studying both coding and non-coding RNAs of the embryo could aid our understanding of the causes and/or effects of the physiological processes accompanying the decline including the differential expression of sub-cellular biomarkers indicative of various diseases. The current study is a post-hoc analysis of the expression of trophectoderm RNA data derived from a previous high throughput study. Its main aim is to determine the characteristics and potential functionalities that characterize long non-coding RNAs. As reported previously, a maternal age-related component is potentially implicated in implantation success. Trophectoderm samples representing the full range of maternal reproductive ages were considered in relation to embryonic implantation potential, trophectoderm transcriptome dynamics and reproductive maternal age. The long non-coding RNA (lncRNA) biomarkers identified here are consistent with the activities of embryo-endometrial crosstalk, developmental competency and implantation and share common characteristics with markers of neoplasia/cancer invasion. Corresponding genes for expressed lncRNAs were more active in the blastocysts of younger women are associated with metabolic pathways including cholesterol biosynthesis and steroidogenesis.

In vitro effects of single and binary mixtures of regulated mycotoxins and persistent organochloride pesticides on steroid hormone production in MA-10 Leydig cell line.

Epidemiological studies have shown strong deterioration in male reproductive health globally due to compromised testosterone production leading to altered spermatogenesis and poor sperm quality. However, the effects and mechanisms through which mycotoxins and persistent organochloride pesticides contribute to poor reproductive health in males remain unclear. The effects of single and binary combinations of ochratoxin A, deoxynivalenol, zearalenone, alpha-zearalenol, beta-zearalenol and 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane on testicular steroidogenesis were evaluated using the MA-10 Leydig cell line after 48 h of exposure. Zearalenone exposure, especially at 16 µM, had a stimulatory effect on progesterone secretion (4.7 ±â€¯0.48 ng/mL compared to 0.60 ±â€¯0.07 ng/mL in control), but inhibited testosterone production after 48 h compared to the solvent control. Ochratoxin A treatment significantly increased both progesterone and testosterone levels. Combination of alpha-zearalenol with beta-zearalenol showed a synergistic stimulation of progesterone hormone level at 1 and 8 µM. The results presented here show that the MA-10 Leydig cell line is a useful model for assessing the effects of xenoestrogens on testicular steroidogenesis. In addition, the inhibitory effects of zearalenone, alpha-zearalenol and beta-zearalenol on testosterone production was enhanced by co-exposure with 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane, further compounding the threat posed by these mycotoxins to male reproductive health.

Can trophectoderm RNA analysis predict human blastocyst competency?

A systematic review of the literature showed that trophectoderm biopsy could assist in the selection of healthy embryos for uterine transfer without affecting implantation rates. However, previous studies attempting to establish the relationship between trophectoderm gene expression profiles and implantation competency using either microarrays or RNA sequencing strategies, were not sufficiently optimized to handle the exceptionally low RNA inputs available from biopsied material. In this pilot study, we report that differential gene expression in human trophectoderm biopsies assayed by an ultra-sensitive next generation RNA sequencing strategy could predict blastocyst implantation competence. RNA expression profiles from isolated human trophectoderm cells were analysed with established clinical pregnancy being the primary endpoint. Following RNA sequencing, a total of 47 transcripts were found to be significantly differentially expressed between the trophectoderm cells from successfully implanted (competent) versus unsuccessful (incompetent) blastocysts. Of these, 36 transcripts were significantly down-regulated in the incompetent blastocysts, including Hydroxysteroid 17-Beta Dehydrogenase 1 (HSD17B1) and Cytochrome P450 Family 11 Subfamily A Member 1 (CYP11A1), while the remaining 11 transcripts were significantly up-regulated, including BCL2 Antagonist/Killer 1 (BAK1) and KH Domain Containing 1 Pseudogene 1 (KHDC1P1) of which the latter was always detected in the incompetent and absent in all competent blastocysts. Ontological analysis of differentially expressed RNAs revealed pathways involved in steroidogenic processes with high confidence. Novel differentially expressed transcripts were also noted by reference to a de novo sequence assembly. The selection of the blastocyst with the best potential to support full-term pregnancy following single embryo transfer could reduce the need for multiple treatment cycles and embryo transfers. The main limitation was the low sample size (N = 8). Despite this shortcoming, the pilot suggests that trophectoderm biopsy could assist with the selection of healthy embryos for embryo transfer. A larger cohort of samples is needed to confirm these findings. Abbreviations: AMA: advanced maternal age; ART: assisted reproductive technology; CP: clinical pregnancy; DE: differential expression; FDR: false discovery rate; IVF: in vitro fertilization; LD PCR: long distance PCR; qRT-PCR: quantitative real-time PCR; SET: single embryo transfer; TE: trophectoderm.

The effect of individual and mixtures of mycotoxins and persistent organochloride pesticides on oestrogen receptor transcriptional activation using in vitro reporter gene assays.

The mycotoxins zearalenone (ZEN) and alpha-zearalenone (α-ZOL), which are common contaminants of agri-food products, are known for their oestrogenic potential. In addition to mycotoxins, food may also contain pesticides with oestrogenic properties such as 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (p,p'-DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE), raising the question on the potential effects of individual and combinations of these xeno-oestrogens on the action of natural oestrogens. The present study employed a mammalian reporter gene assay to assess the effects individual and binary combinations of these environmental and food-borne contaminants on oestrogen nuclear receptor (ER) transactivation. As expected, α-ZOL and ZEN exhibited the strongest oestrogenic potency (EC50: 0.27 ±â€¯0.121 nM and 1.32 ±â€¯0.0956 nM, respectively) whereas p,p'-DDT and p,p'-DDE had weak ER agonistic activity with the maximal response of 28.70 ±â€¯2.97% and 18.65 ±â€¯1.77%, respectively. Concurrent treatment of the mycotoxins and/or pesticides, individually or in binary combination, with 17ß-oestradiol (E2) showed either additive, synergistic or antagonistic interactive effects on E2-mediated ER response, depending on the combination ratios, the concentration range of xeno-oestrogens, and the concentration of E2. This study highlights the importance of assessing the mixture effects of chemical contaminants in risk assessment, especially in the area of reproductive and developmental toxicity.

Toxicological effects of regulated mycotoxins and persistent organochloride pesticides: In vitro cytotoxic assessment of single and defined mixtures on MA-10 murine Leydig cell line.

Epidemiological studies show that there is global decline in male fertility primarily as a result of poor sperm quality and this is attributed to exposure to endocrine disrupting chemicals (EDCs) in the environment, food and pharmaceutical products, including mycotoxins and pesticides. The Leydig cells in the male testes are responsible for producing androgens, hormones that play major roles in male development and reproductive function. Therefore, any toxin that affects the function and morphology of the Leydig cells may result in sub-fertility or infertility. The cytotoxic effects of single and binary mixtures of aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), alpha-zearalenol (α-ZOL), beta-zearalenol (ß-ZOL), 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (p,p'-DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE) on a model cell line, the MA-10 Leydig cells, were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-dipenyltetrazolium bromide (MTT) assay after 48h of exposure. With single toxin treatment at doses between 0.1µM and 64µM for 48h, DON was the most cytotoxic to MA-10 cells with a half maximal inhibitory concentration (IC50) value of 12.3µM followed by α-ZOL (IC50: 28µM) and OTA (IC50: 30µM) while the IC50 of AFB1, p,p'-DDT and p,p'-DDE were above the highest concentration tested (64µM). Co-exposure with p,p'-DDT or p,p'-DDE enhanced the toxicity of DON, OTA and ZEN to MA-10 Leydig cells, particularly at higher concentrations. This highlights the possible adverse effects on male reproductive health following co-exposure to these toxins.

Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos.

PURPOSE: Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. METHODS: Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. RESULTS: GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. CONCLUSIONS: To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos.

Mutations causing familial biparental hydatidiform mole implicate c6orf221 as a possible regulator of genomic imprinting in the human oocyte.

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.

Quantitative analysis of DNA methylation of imprinted genes in single human blastocysts by pyrosequencing.

We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.