Preparation of Anti-Zearalenone IgY and Development of an Indirect Competitive ELISA Method for the Measurement of Zearalenone in Post-Fermented Tea.

Post-fermented tea (PFT) is one of the most commonly consumed beverages worldwide. Rapid microbial growth and significant changes in the microbial composition of PFT during processing and storage pose a potential risk of contamination with mycotoxins such as zearalenone (ZEN). Screening for ZEN contamination in a simple, rapid, and inexpensive manner is required to ensure that PFT is safe for consumption. To monitor ZEN in PFT, ZEN was conjugated with bovine serum albumin to prepare egg yolk immunoglobulins (IgY). A specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on IgY was developed and validated. ZEN was extracted with acetonitrile and water (50:50, v/v) containing 5% acetic acid and purified using a mixture of primary and secondary amines and graphitized carbon black to remove matrix interference from the PFT samples. Under optimal conditions, the linear range of this assay was 13.8-508.9 ng mL-1, the limit of detection was 9.3 ng mL-1, and the half-maximal inhibitory concentration was 83.8 ng mL-1. Cross-reactivity was negligible, and the assay was specific for ZEN-related molecules. The recovery rate of ZEN in the control blanks of PFT samples spiked with a defined concentration of ZEN of 89.5% to 98.0%. The recovery and accuracy of the method were qualified for PFT matrices. No significant differences were evident between the results of the actual PFT samples analyzed by high-performance liquid chromatography and ic-ELISA. The collective data indicate that the developed ic-ELISA can be used for the rapid and simple detection of ZEN in PFT products.

B-Type Fumonisins in Post-Fermented Tea: Occurrence and Consumer Dietary Exposure in Guangxi, China.

Post-fermented tea (PFT), a commonly consumed beverage worldwide, is characterized by the rapid growth of its microbial groups and the substantial changes they undergo. Consequently, PFT may contain mycotoxins such as B-type fumonisins (FBs). This study aimed to assess the intake of FBs through the consumption of PFT among consumers in Guangxi, China. A novel quantitative method using high-performance liquid chromatography-mass spectrometry was used to determine the FB concentration in PFT products. Additionally, a PFT consumption survey was conducted using a face-to-face questionnaire, recording their body weight and PFT consumption patterns based on a three-day dietary recall method. Finally, hazard index was calculated to estimate the health risk of FBs from the consumption of PFT products in Guangxi. The results revealed that the occurrence of FBs in PFT was 20% (24/120), with a concentration ranging from 2.14 to 18.28 µg/kg. The results of the survey showed that the average daily consumption of PFT by consumers was 9.19 ± 11.14 g. The deterministic risk assessment revealed that only 0.026% of the provisional maximum tolerable daily intake of FBs was consumed through PFT, indicating that FB contamination in PFT is not a public health risk.

Novel lncRNA-HZ04 promotes BPDE-induced human trophoblast cell apoptosis and miscarriage by upregulating IP3 R1 /CaMKII/SGCB pathway by competitively binding with miR-hz04.

Normal pregnancy is essential for human reproduction. However, BaP (benzo(a)pyrene) and its metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) could cause dysfunctions of human trophoblast cells and might further induce miscarriage. Yet, the underlying mechanisms remain largely unknown. Herein, we identified a novel upregulated lnc-HZ04 and a novel downregulated miR-hz04 in villous tissues of unexplained recurrent miscarriage (RM) relative to those in healthy control tissues and also in BPDE-treated human trophoblast cells. Lnc-HZ04 directly and specifically bound with miR-hz04, diminished the reduction effects of miR-hz04 on IP3 R1 mRNA expression level and on IP3 R1 mRNA stability, and then activated the Ca2+ -mediated IP3 R1 /p-CaMKII/SGCB pathway, which further promoted trophoblast cell apoptosis. The miR-hz04 target site on lnc-HZ04 played crucial roles in these regulations. In normal trophoblast, relatively less lnc-HZ04 and more miR-hz04 suppressed this apoptosis pathway and gave normal pregnancy. After exposure to BPDE or in RM tissues, p53 was upregulated, which might promote p53-mediated lnc-HZ04 transcription. Relatively more lnc-HZ04 and less miR-hz04 activated this apoptosis pathway and might further induce miscarriage. BaP could also induce mice miscarriage by upregulating its corresponding murine apoptosis pathway. Therefore, BPDE-induced apoptosis of human trophoblast cells was associated with the occurrence of miscarriage. This work discovered the regulation roles of lnc-HZ04 and miR-hz04 and provided scientific and clinical understanding of the occurrence of unexplained miscarriage.

Egg Yolk Immunoglobulin Supplementation Prevents Rat Liver from Aflatoxin B1-Induced Oxidative Damage and Genotoxicity.

Egg yolk immunoglobulins (IgY), as nutraceutical supplement for therapeutic or prophylactic intervention, have been extensively studied. The effects of IgY on small molecular toxin-induced toxicity in animals are unclear. In the present study, the protection of highly purified and specific anti-AFB1 IgY against AFB1-induced genotoxicity and oxidative damage on the rat liver model were investigated. Our results revealed that AFB1 induced significant oxidative damage markers, as well as AFB1-induced protein expression in antioxidant, pro- and antiapoptosis processes in rat liver. These effects could be significantly inhibited by cogavage with anti-AFB1 IgY in a dose-dependent manner. However, anti-AFB1 IgY did not significantly induce hepatic CAT and SOD1. To explore mechanisms, metabolite experiments were established to evaluate the influence of anti-AFB1 IgY on the absorption of AFB1 in rats. Middle and high doses of anti-AFB1 IgY reduced hepatic AFB1-DNA adducts by 43.3% and 52.9%, AFB1- N7-guanine urinary adducts by 19.6% and 34.4%, and AFB1-albumin adducts by 10.5% and 21.1%, respectively. The feces of high dose anti-AFB1 IgY cogavaged rats contained approximately 2-fold higher AFB1 equivalents at 3-6 h after ingestion than AFB1 group feces, indicating IgY inhibited AFB1 uptake. These results had provided insight that anti-AFB1 IgY could prevent animal organs from damage caused by AFB1 and will be beneficial for the application of detoxification antibody as a supplement in food.

IgY Reduces AFB1-Induced Cytotoxicity, Cellular Dysfunction, and Genotoxicity in Human L-02 Hepatocytes and Swan 71 Trophoblasts.

Aflatoxin B1 (AFB1) causes hepatotoxic, genotoxic, and immunotoxic effects in a variety of species. Although various neutralizing agents of AFB1 toxicity have been studied, the egg yolk immunoglobulin (IgY) detoxification of small molecular toxins and the mechanisms underlying such effects have not yet been reported. In this investigation, anti-AFB1 IgY against AFB1 was successfully raised, and a competitive indirect enzyme-linked immunosorbent assay was established with a sensitive half-maximal inhibitory concentration (IC50, 2.4 ng/mL) and dynamic working range (0.13-43.0 ng/mL). The anti-AFB1 IgY obtained reduced AFB1-induced cytotoxicity, cellular dysfunction, and genotoxicity by protecting cells against apoptotic body formation and DNA strand breaks, preventing G2/M phase cell cycle arrest, reducing AFB1-DNA adduct and reactive oxygen species production and maintaining cell migration and invasion and the mitochondrial membrane potential. Anti-AFB1 IgY significantly inhibited the AFB1-induced expression of proteins related to antioxidative, pro-apoptotic, and antiapoptotic processes in a strong dose-dependent manner. These experiments demonstrated that the anti-AFB1 IgY-bound AFB1 could not enter cells. This is the first time that IgY has been found to reduce the effects of small molecular toxins, which will be beneficial for the development of antibodies as detoxication agents.