Effects Of Aflatoxin B1 Subchronic Exposure On Oxidative And Genetic Damage In Male Mice And Its Mechanism

Aflatoxin B1(aflatoxin B1,AFB1)is one of the strongest toxicity and the biggest threat to human in known mycotoxins.In addition it can also pollute the peanut,soybean grain and oil crops.Its indirectly pose a threat to all kinds of processed food and livestock through the food chain.Due to its strong carcinogenicity,and its chemical structure is stable and difficult to be decomposed under high temperature,AFB1 exposure will eventually lead to the occurrence of various types of acute or subchronic poisoning.Although the carcinogenic effect of AFB1 and its related mechanism has been confirmed,but its research on the organisms causing oxidative and genetic damage and its mechanism is not very clear,this is not conducive to the in-depth study of the mechanism of toxicity of aflatoxins.In addition,it also caused some obstacles on the establishment of AFB1 defense system.In this study,we studied the oxidative,genetic toxic effects and related molecular mechanism of AFB1 in order to provide a reference for the toxicity of AFB1,elucidate the toxicity mechanism of other categories of aflatoxin,and also provide more reference for the degradation of aflatoxin detoxification or defense in vivo poisoning.In this study,a total of 40 male mice for 50 consecutive days in 3 different doses(0.09375,0.375,1.5mg/kg b.w./day)AFB1 exposure.In order to evaluate the oxidative damage and the molecular mechanism of AFB1 caused,we investigated the damage index,liver cell apoptosis and its related signaling pathways in the oxidation of serum and organs.In order to evaluate the genetic toxicity and molecular mechanism of DNA,the DNA strand breakage,cross-linking,cell micronucleus and sperm abnormality were examined.Then,the interaction mechanism between AFB1 and DNA was studied in vitro,and it was associated with genotoxicity.The specific research contents and results are as follows:(1)after AFB1 exposure for about 50 days,AFB1 can cause obvious organ damage and increase of serum ALT and AST levels in mice.The weight of mice in each group decreased with the increase of the dose,and the weight of the three organs of liver,kidney and testis also showed a decreased trend,indicating that the three organs were affected by AFB1 exposure.The pathological damage caused by AFB1 was as follows: the inflammation was the main factor at low dose,and the local necrosis,swelling of the glomerulus and necrosis of tubular epithelial cells were observed at high dose.(2)AFB1 can produce significant oxidative damage to the mouse liver,under the subchronic conditions.Both different doses of groups can lead to elevated ROS in liver.The levels of SOD and GSH was declined in the middle and high dose groups showed that the antioxidant system was damaged and the anti-oxidative mechanism could not be started to remove excess ROS.However,in the low dose group,the liver was able to activate its antioxidant mechanism,which was prevented from being damaged by excessive ROS.The levels of lipids,proteins and DNA in the liver of middle and high dose groups were all attacked by ROS,which caused oxidative damage and the body was in oxidative stress.However,AFB1 did not produce significant macromolecular substance in low dose group influences.(3)The oxidative damage induced by AFB1 is not only a result of cell necrosis,but also a possible way of apoptosis,and the apoptosis rate increases with the increase of AFB1 concentration.At low dose,the effect of AFB1 on cells was mainly inflammation and local necrosis,while in the large dose,apoptosis was the main factor.The molecular mechanism of AFB1 mediated apoptosis may be related to the activation of Bax,Caspase-3,P53 protein and the expression of Bcl-2 protein.Molecular mechanism of apoptosis in oxidative injury induced by subchronic exposure to AFB1.This suggests that,if we can start from the point of view of apoptosis and have a better regulation,it will be possible provide a new way to curb the oxidative toxicity of AFB1.(4)AFB1 exposure in subchronic conditions can lead to DNA strand breaks,DNA-DNA cross-linking and DNA-protein cross-linking in male mice.At the same time,it also lead to increased sperm deformity,micronucleus formation and chromosome damage.The five indexes except DNA-DNA cross-linking,the rest indexes increase with the dose of AFB1 increased,and has a dose effect relationship,suggesting that AFB1 has genetic toxicity not only on the somatic cells,but also on germ cells,and could produce mutagenic effect.(5)When the larger dose of AFB1 was exposed to mice(1.5mg/kg),the DNA-DNA crosslinking rate did not increase with the increase of AFB1 concentration.This result indicates that the proportion of DNA-DNA cross-linking in the various DNA damage forms is reduced when the AFB1 concentration increases to a certain extent(1.5 mg / kg),suggesting that other forms of DNA damage(e.g.,DNA strand breaks,DNA-protein cross-linking)increased the proportion.The mechanism of genetic toxicity of AFB1 may be caused by a large number of free radicals such as ROS,which are caused by oxidative stress,directly attack DNA,or ROS mediated oxidative stress indirectly leads to the formation of DNA oxidative damage.(6)AFB1 does not need to be metabolized and can interact directly with DNA to form binary complexes.At the same time,DNA can obviously quench the intrinsic fluorescence of AFB1,and the type of quenching is static and dynamic quenching.The binding constant is 4.12×103,which is far less than the binding constant.The interaction force between DNA and AFB1 are mainly hydrogen bond and hydrophobic interaction.The binding mode between AFB1 and DNA is bonded to the grooving.The binding of AFB1 leads to a slight change in the conformation of DNA,but has no significant effect on its conformation.Molecular docking results show that the combination of AFB1 and DNA is located in the small groove region,and the O6 and O3 atoms of AFB1 and DA-17,DG16 two bases form three strong hydrogen bonds.This suggests that there may be a new mode of interaction in inducing DNA damage by AFB1,which may be related to toxic mechanism of AFB1.In conclusion,subchronic AFB1 exposure to the mouse oxidation,genetic system caused a certain degree of damage,and its mechanism of action with the body in the oxidative stress state,a large number of ROS generation.The development of this research has a guiding significance for the carcinogenic mechanism and related drugs.