ZEN Interferes With The Toxic Effects Of AFB₁ On Mammary Gland,ovary,liver And Spleen In Rats And Its Mechanism

Aflatoxin B₁（AFB₁）,as the strongest known carcinogen in nature,can seriously affect the performance and health of livestock and poultry.It can metabolise into Aflatoxin M₁（AFM₁）in the liver,which can enter the milk through the mammary gland,causing food safety problems.Both AFB₁ and AFM₁ may damage the mammary gland and affect the milk quality.Zearalenone（ZEN）is a mycotoxin that can disrupt hormone homeostasis and metabolic activity and may change the sensitivity of animals to exogenous harmful substances.ZEN often contaminates feed together with AFB₁,and pregnant and lactating mammals are particularly vulnerable to the effects of ZEN.In this study,the effects of AFB₁and AFM₁ on mammary gland cells and their mechanisms were studied by cell assays.The toxicity of AFB₁ on mammary glands and ovaries by pregnant and lactating rats,as well as the effect and mechanism of ZEN on this toxicity,were studied.The results of this study provide theoretical guidance and a scientific basis for evaluating the effects of mycotoxins on pregnant and lactating mammals and formulating relevant feed hygiene measures.The research is based on the following three aspects:1.Effects of AFB₁ and AFM₁ on breast cells and their mechanismsBovine mammary epithelial cells were divided into six groups:control group,2 mg/L AFB₁ group,10 mg/L AFB₁ group,2 mg/L AFM₁ group,10 mg/L AFM₁ group and 2 mg/L AFB₁+2 mg/L AFM₁ group.After the cells had been treated with the corresponding toxin level for 24 h,the effects of the AFB₁and AFM₁ on cell survival rate,cell apoptosis,cell cycle,mitochondrial membrane potential,antioxidant capacity,m⁶A methylation level in total RNA and the conversion level of AFB₁ into AFM₁ were detected,and the effects of the aflatoxin on gene expression were analysed by transcriptomics.The main results are as follows:1.1 Both AFB₁ and AFM₁ decreased the cell survival rate,the proportion of cells in the division stage,and the proportion of cells with abnormal mitochondrial membrane potential,and the reduction of AFB₁ is more severe than AFM₁.Both AFB₁ and AFM₁decreased the antioxidant capacity.AFM₁ was not detected after AFB₁ was cultured with breast cells.Both AFB₁ and AFM₁ showed strong cytotoxicity to breast cells,and AFB₁was more toxic than AFM₁ at the same dose.However,the breast cells could not convert AFB₁ into AFM₁.Compared with 2 mg/L AFB₁ or AFM₁ alone,2 mg/L AFB₁+2 mg/L AFM₁ group caused more severe cell death,cell cycle arrest and apoptosis,suggesting that AFB₁ and AFM₁ had an additive effect on the toxicity to breast cells.1.2 Transcriptomics’result found that AFB₁ and AFM₁ caused changes in the expression levels of many cytotoxic genes related to cell proliferation,apoptosis,cell cycle,tight intercellular connection and oxidative stress and reduced the methylation level of RNA m⁶A in cells.These results suggest that AFB₁ and AFM₁ have a wide range of dose-dependent effects on gene expression in mammary gland cells,and they may exert their toxic effects through epigenetic gene modification.The number of differential genes induced in the AFB₁ group was significantly higher than that in the AFM₁ group,indicating that AFB₁ had a greater effect on gene expression in mammary gland cells.Compared with2 mg/L AFB₁ or AFM₁ alone,the 2 mg/L AFB₁+2 mg/L AFM₁ group showed more severe cell damage and more changes in related gene expression,suggesting that AFB₁ and AFM₁had an additive effect on the toxicity to breast cells.2.Study of AFB₁-induced toxicity to rat mammary glands and ovaries and the effect of ZEN on AFB₁-induced toxicityForty pregnant Sprague-Dawley（SD）rats were randomly divided into four treatment groups with ten rats in each group,and each was kept in a single cage.Each group was fed different diets:Control group（fed a basal diet）,AFB₁ group（fed a basal diet+2 mg/kg AFB₁）,AFB₁+1 ZEN group（fed a basal diet+2 mg/kg AFB₁+1 mg/kg ZEN）and AFB₁+10ZEN group（fed a basal diet+2 mg/kg AFB₁+10 mg/kg ZEN）.The experiment lasted for the gestation period and the lactation period.Growth performance parameters such as average body weight,average daily gain and average daily feed intake as well as reproductive performance parameters,such as litter size,milk yield and litter weight of female rats,were recorded.At 35 d,serum,mammary glands and ovaries of female rats were collected for blood biochemical analysis,RT-q PCR,metabolomics and transcriptomic analysis to explore the mechanism of AFB₁-induced toxicity to rat mammary glands and ovaries and the effect of ZEN on AFB₁-induced toxicity.The main experimental results are as follows:2.1 Compared with the control group,the AFB₁ group showed a significantly reduced average body weight,average daily gain and average daily feed intake of female rats during pregnancy and lactation（P<0.05）.The average weight of offspring was decreased by24.3%,but the number of offspring was increased by 2.4 on average.The levels of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,alkaline phosphatase（ALP）,blood urea nitrogen（BUN）and glucose in the AFB₁ group were significantly increased by38.3%,70.6%,78.5%,22.0%and 17.9%,respectively（P<0.05）.Total protein（TP）,albumin（ALB）,cholesterol（CHOL）and triglyceride（TG）levels were significantly decreased by 15.6%,13.3%,14.7%and 9.0%,respectively（P<0.05）.Compared with the AFB₁ group,the AFB₁+1 ZEN group showed mitigated effects on feed intake,body weight and serum biochemical indices,whereas the AFB₁+10 ZEN group showed enhanced effects on feed intake,body weight and serum biochemical indices.The results showed that AFB₁decreased the growth and reproductive performance of gestation and lactation rats and changed the blood biochemical indices.Further,ZEN affected the negative impacts of AFB₁ on growth performance,reproductive performance and blood biochemical indices of rats.The effect of ZEN on AFB₁ showed that it partially antagonized the harm of AFB₁ at low dose and enhanced the harm of AFB₁ at high dose.2.2 Compared with the control group,AFB₁group change the histological morphology of the mammary gland and ovary,increased the contents of cytokines（IL-1β,IL-6 and TNF-α）,8-hydroxydeoxyguanosine（8-OHd G）in breast and ovary（P<0.05）,and three breast cancer markers in serum.AFB₁group reduced by 12.2%on the 13th day of milk production,decreased the level of PRL and E₂in serum,mammary gland and ovary（P<0.05）.In the breast and the ovary,the AFB₁ group showed a significant（P<0.05）effect for ER stress（GRP78,ATF4,ATF6 and CHOP）,heat shock protein（Hsp27,Hsp70 and Hsp90）,apoptosis（p38,p53,Bax,and Bcl-2）,inflammation（Nlrp3,IL-1β,IL-6,and TNF-α）and sex hormone receptors（Esr1 and Prlr）.The level was significantly Compared with the AFB₁ group,the AFB₁+1 ZEN group showed reduced effects on lactation volume,inflammatory factors,8-OHd G and related genes,whereas in the AFB₁+10 ZEN group,the effects on the expressions of lactation yield,inflammatory factors,8-OHd G and related genes were enhanced.Both the AFB₁+1 ZEN group and the AFB₁+10 ZEN group showed increased levels of breast cancer markers and decreased levels of E₂ and PRL.Based on the results,AFB₁ damaged the tissue morphology of the breast and ovary,induced ER stress and inflammation and reduced the level of sex hormones.The effect of ZEN on AFB₁showed that it partially antagonized the harm of AFB₁ at low dose and enhanced the harm of AFB₁ at high dose.ZEN affected the damage of AFB₁ to the breast and ovary,further disrupting sex hormone levels and increasing the risk of breast cancer.2.3 Mammary gland metabolomics found that in group AFB₁,mainly amino acid and glucose metabolism were affected.The AFB₁+1 ZEN group had the most abundant metabolites,whereas in the AFB₁+10 ZEN group,mainly lipid metabolism was affected.Some metabolic markers of ZEN on AFB₁ toxicity were found by ROC analysis.In the ovarian transcriptome,AFB₁ affects the expression of many genes related to steroidal compounds,lipid metabolism and inflammatory responses.Some key node genes and abnormally expressed genes,such as SFRP2,Msx1 and Hoxd10,which may be involved in the effect of ZEN on AFB₁ toxicity,were identified by the network diagram and the heatmap of fold change.Compared with the control group,the AFB₁ group showed 37.5%lower levels of RNA m⁶A methylation in the mammary gland.The expressions of METTL3and METTL14 were significantly down-regulated,whereas those of FTO,ALKHB5 and YTHDF1 were significantly up-regulated（P<0.05）.The results showed that AFB₁ affected the metabolic activity in the mammary gland as well as gene expression and epigenetic modification in the ovary,whereas ZEN had an impact on the effect of AFB₁ on metabolites and epigenetic modification.The effect of ZEN on AFB₁ showed that it partially antagonized the harm of AFB₁ at low dose and enhanced the harm of AFB₁ at high dose.Further,ZEN may affect the toxicity of AFB₁ to mammary glands and ovaries through influencing cell metabolic activities,RNA m⁶A methylation,key node genes,and abnormally expressed genes.3.Study of AFB₁-induced toxicity to rat liver and spleen and the effect of ZEN on AFB₁-induced toxicityThe experimental design was the same as before,and the rats were the same batch of animals.The livers of the dams and offspring and the spleens of the offspring were collected,with the aim to study the toxicity of AFB₁ on liver and spleen of rats and the effect and mechanism of ZEN on AFB₁ toxicity.The main experimental results are as follows:3.1 Compared with the control group,the levels of antioxidant capacity in the livers of the dams and their offspring in group AFB₁ were significantly lower（P<0.05）.The AFB₁ group also showed significantly increased the levels of cytokines（IL-1β,IL-6,and TNF-α）and 8-OHd G by 1.6–3.5 and 8.8 times in serum,respectively（P<0.05）.Regarding the liver and spleen of the rat dams,in the AFB₁ group,we observed a significantly impacted expression of genes related to ER stress,heat shock proteins,apoptosis and inflammatory responses and a significantly decreased level of RNA m⁶A methylation（P<0.05）.The expressions of METTL3 and METTL14 were down-regulated by 31.7%,the expressions of FTO,ALKHB5 and YTHDF1 were significantly up-regulated in liver（P<0.05）.Compared with the AFB₁ group,the AFB₁+1 ZEN group showed reduced antioxidant capacity,inflammatory factors and epigenetic inheritance,whereas in the AFB₁+10 ZEN,these parameters were enhanced.The results showed that AFB₁ induced oxidative stress and inflammation in the liver and spleen and decreased antioxidant capacity and RNA m⁶A methylation level in the liver,whereas ZEN influenced the toxicity of AFB₁ to liver and spleen.The effect of ZEN on AFB₁ showed that it partially antagonized the harm of AFB₁at low dose and enhanced the harm of AFB₁ at high dose.3.2 Compared with the control group,the AFB₁ group showed a significantly（P<0.05）up-regulated expression of genes related to phaseⅠmetabolism（CYP1A2,CYP1B1,CYP2B1,CYP2E1）in the liver of the rat dams and a significantly down-regulated（P<0.05）expression ofⅡdetoxification-related genes（GSMT1,CAT,Nrf2,Keap1）.Compared with the AFB1 group,the expression levels of CYP1B1,CYP2B1,GSMT1 and Nrf2 in the AFB₁+1 ZEN group were significantly down-regulated（P<0.05）.In the AFB₁+10 ZEN group,the expression of related to phaseⅠmetabolism was significantly up-regulated,whereas that of genes related to phaseⅡdetoxification（GSMT1 and Nrf2）was significantly down-regulated（P<0.05）.In the AFB₁+1 ZEN group and the AFB₁+10 ZEN group,the AFM₁ levels in maternal serum,neonatal serum and the mammary gland were significantly increased by 16.9%–45.4%（P<0.05）.The results showed that AFB₁ affected the expression of genes related to phaseⅠmetabolism and phaseⅡdetoxification in the liver of the rat dams.Further,ZEN may affect the transformation of AFB₁ into AFM₁ by enhancing the expression of genes related to phaseⅠmetabolism and phaseⅡdetoxification.4.Conclusions4.1 Both AFB₁ and AFM₁ have a strong cytotoxicity and accumulative effect,can induce cell necrosis,apoptosis,cycle arrest and oxidative stress and can decrease the antioxidant capacity of mammary gland cells.4.2 The cytotoxicity of AFB₁ is stronger than that of AFM₁,and the mechanism of action may be to regulate the expression of downstream genes by influencing epigenetic modification,resulting in cytotoxicity.4.3 Exposure to AFB₁ during gestation and lactation can inhibit the growth of the rat dams and their offspring,cause endoplasmic reticulum stress,cell apoptosis and inflammatory reactions in the mammary gland,ovary,liver,and spleen.AFB₁can affect the expression of related genes in the ovary,impact metabolic activities in the mammary gland and epigenetic modification and reduce the levels of sex hormones.4.4 ZEN affects the damage of AFB₁ on the mammary gland,ovary,liver,and spleen,and has a dual-effect on AFB₁ toxicity.It enhances the toxicity of AFB₁ at high doses but partially attenuates the damage caused by AFB₁ at low doses.ZEN can enhance the metabolic transformation of AFB₁ to AFM₁,further disrupt sex hormone levels,and increase the risk of breast cancer caused by AFB₁.The mechanism of ZEN affecting AFB₁toxicity may be through various ways such as cell metabolic activities,sex hormone levels,key node genes,and abnormal expression genes.