Study On The Single And Combined Toxicity Of DON And AFB₁ On Intestinal Damage In Mice

Mycotoxins are toxic secondary metabolite produced by organisms of the fungus kingdom under the specific environment which are capable of causing disease and death in both humans and other animals.They are widely existed in the feed and agricultural products and can produce serious damage.Among them,deoxynivalenol（DON）and aflatoxin B₁（AFB₁）are two kinds of mycotoxins which have the highest contamination rates and levels in feed and feedstock.In recent years,animals have caused serious losses due to the ingestion of mycotoxins which contaminated feed as well as seriously hindered the development of livestock and poultry husbandry.Toxicity of mycotoxins and their combined effects has become a topic sought after by scholars from various countries However,reports of intestinal injury in mice have been rarely reported in combination with DON and AFB₁.Thus the aim of this study was to provide a theoretical basis for the combined toxicity of DON and AFB₁ and comparing both single and combined effects of two toxins on the intestinal tract of mice.Ninety-six Kunming male mice aged 18 d were randomly divided into 4 groups.（1）The first group was control group（CG）which were feed daily physiological saline;（2）DON group was given 500μg·kg-1 DON daily;（3）AFB₁ group were feed 200μg·kg-1 AFB₁daily;and（4）DON+AFB₁ group which were fed with 500μg·kg-1 DON+200μg·kg-1AFB₁ daily.The trial period is lasted for 45 d.Six mice were randomly selected from each group on day 0,15th,30th,and 45th of the experiment.Blood was collected from the eyeballs and the serum was separated to determine the total protein（TP）and albumin（ALB）content.At the same time,the necropsies samples were taken from the duodenum and ileum for determination of（1）the contents of TP and ALB;（2）Oxidation and antioxidant indices,the action of glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）and hydroxyl radical（·OH）as well as the content of malondialdehyde（MDA）.（3）Intestinal permeability indexes:diamine oxidase（DAO）activity and interleukin 4（IL-4）levels,complement protein 3（C₃）and complement protein 4（C₄）content.（4）mRNA expression of intestinal tight junction proteins Occludin and ZO-1.On day 0 and 45th of the experiment,fresh feces from the rectum were taken under aseptic conditions for detection of changes in intestinal flora.The results show:1.On the 15th day of the experiment,compared with the CG,the contents of TP and ALB in the DON group were significantly decreased（P<0.01）.GSH-Px activity in the duodenum of the DON group,AFB₁ group and DON+AFB₁ group as well as content of DAO,IL-4,C₃ were significantly decreased（P<0.01）.MDA content and OH activity were also significantly increased（P<0.01）.The mRNA expression of Occludin was significantly reduced in the ileum of AFB₁ group and DON+AFB₁ group（P<0.01）,while the mRNA expression of ZO-1 was also decreased（P>0.05）.Compared with the DON+AFB₁ group,the activities of SOD and OH of DON group and AFB₁ group were significantly increased（P<0.01）,and both GSH-Px action and IL-4 level were considerably decreased（P<0.01）.2.On the 30th day of the experimental trials,during comparison with the CG,SOD activity in the duodenum of DON group,AFB₁ group and DON+AFB₁ group was significantly lower（P<0.01）.MDA content and OH activity were significantly higher（P<0.01）and IL-4 concentration,complement C₃,C₄ content were significantly reduced（P<0.01）.The mRNA expression of Occludin,ZO-1 in ileum of DON group and DON+AFB₁ group as well as the ZO-1 mRNA expression of AFB₁ group were significantly increased（P<0.01）.Compared with the DON+AFB₁ group,the activity of SOD and DAO in DON and AFB₁ group were significantly improved（P<0.01）while MDA content and IL-4 concentration were significantly decreased（P<0.01）.3.On the 45th day of the experiment,during comparison with the CG,the TP content in duodenum of DON group and AFB₁ group as well as DON+AFB₁ group and the ALB content in DON group and DON+AFB₁ group were significantly decreased（P<0.01）.GSH-Px activity in duodenum of AFB₁ and DON+AFB₁ group were significantly increased（P<0.01）as well as SOD action and MDA content in duodenum of DON,AFB₁,andDON+AFB₁ group as well as OH and IL-4 vitality was significantly increased（P<0.01）.The mRNA expression of Occludin was significantly increased in the ileum of DON group and AFB₁ groups as well as DON+AFB₁ group（P<0.01）.Compared with the DON+AFB₁group,SOD activity of DON and AFB₁ groups were significantly increased（P<0.01）,IL-4concentration,complement C₃ content and the mRNA expression of Occludin in intestinal tract were significantly decreased（P<0.01）.In the intestinal micro flora,compared with the CG,the abundances of the Bacteroidetes,Deferribacteres,Cyanobacteria and Tenericutes of the DON,AFB₁ and DON+AFB₁ group were significantly decreased（P<0.01）whilethe abundances of the Firmicutes were significantly increased（P<0.01）.Compared with the DON+AFB₁ group,the Deferribacteres and Bacteroidetes loads of DON group and AFB₁ group were significantly increased（P<0.01）but the abundances of Actinobacteria were found statistically significantly decreased（P<0.05）.In conclusion,DON,AFB₁ and DON+AFB₁ can reduce the contents of serum and intestinal proteins and antioxidant function in the intestinal tract of mice.It can also decrease the tight junction protein Occludin and ZO-1 mRNA in the intestine as well as increase intestinal permeability and the exhibits obvious changes in the abundance of intestinal flora.It has also been found that the joint toxicity of DON and AFB₁ is greater than that of single toxicity which has a synergistic effect.