Effects Of Aflatoxin B₁ On Growth Performance And The Expression Of Hepatic Glutathione S-transferase In Broilers

Aflatoxins（AF） are produced as secondary metabolites by the fungus Aspergillus flavus, A.parasiticus, and A. nominus.,which are commonly found in animal feed raw materials and foodstuff. In a variety of aflatoxins, particularly the amount of aflatoxin B₁ is the largest and the toxicity is the strongest, it is carcinogenic, teratogenic and mutagenic.The AFB₁ is toxic to a wide range of animal species. AFB₁ is principally an hepatotoxin and hepatocarcinogen to poultry, but it causes a myriad of other effects either directly or indirectly associated with this toxicity: immunosuppresion, reduced growth rate,lowered egg production, reduced reproductivity, reduced feed utilization and efficiency and even death.Glutathione S-transferase enzymes（GSTs） are widely distributed in the body of a group of various biological multifunctional isoenzymes, which catalyzes AFB₁-8, 9-epoxide（AFBO） and reduced mercapto glutathione conjugate, to increase its hydrophobicity so that it is easy to pass through the cell membrane and excreted after being decomposed, so as to achieve the purpose of detoxification. Glutathione S-transferase enzyme plays a decisive role in the detoxification metabolism of AFB₁ in the body, but not every GST isozyme plays the same important role. Different GSTs have different substrate specificities. Currently,researchers have found that GSTs isoenzyme that is most affinity for AFBO in some mammals. There are many types of glutathione S-transferase isoenzymes in poultry, but the researchs on their affinity to AFBO are few. So it is very necessary to find out the role of all kinds of glutathione S-transferase isoenzymes in AFB₁ detoxification metabolism of poultry.In order to explore the toxic effects of aflatoxin B₁ on poultry and the detoxification function of hepatic glutathione s-transferase genes in poultry,the test has studied the effects of different concentrations of AFB₁ added in a normal diet on the growth performance, the activity of hepatic glutathione S-transferase, the gene expression of eight kinds of hepatic glutathione S-transferase and the protein expression of four kinds of typical hepatic glutathione S-transferase enzyme of male broilers.Test using 1-day-old male Luyuan chicken, pre-fed to 7-day-old,were randomly divided into three groups, the control group was fed the basal diet, the experimental group were added 1mg / kg AFB₁,2mg / kg AFB₁, feeding to 28- day-old, every seven days to observe the growth of broilers and determine the activity of hepatic GSTs which catalyzed substrate CDNB（2,4- Dinitrochlorobenzene）, DCNB（1,2-dichloro-4- nitrobenzene）, p-NBC（4- nitro-benzyl chloride）. using RT-PCR to investigate the mRNA expression of different hepatic GSTs in14-day-old, 21-day-old and 28-day-old broilers :GSTA2, GSTA-CL3, GSTA3, GSTA1, GSTT1, GSTZ1, GSTK1 GSTO1; use Western blotting to investigate the protein expression of three kinds of typical hepatic GSTs in 28-day-old broilers: GSTA2, GSTT1 and GSTO1.The main contents and conclusions are as follows:（1） the effects of aflatoxin B₁ on the growth and the activity of hepatic GSTs in broilers: Both the 1mg / kg AFB₁ group and the 2mg / kg AFB₁ group,the weight and average daily gain（ADG）of 28-day-old broilers were significantly lower（P <0.05）, The average daily feed intake between 21 day-old and 28-day-old was significantly lower（P <0.05）,the feed conversion ratio between 21 day-old and 28-day-old was significantly higher（P <0.05）.In the 1mg / kg AFB₁ group,the hepatic GSTs enzymatic activity to DCNB of 21-day-old chickens was significantly decreased（P <0.05）. In the 2mg / kg AFB₁ group, the hepatic GSTs enzymatic activity to DCNB,CDNB of 21-day-old chickens were significantly decreased（P <0.05）. Both the 1mg / kgAFB₁ group and the 2mg / kgAFB₁ group,the hepatic GSTs enzymatic activity to DCNB,CDNB and p-NBC of 28-day-old chickens were significantly decreased（P <0.001）. This study demonstrates that AFB₁ exposure at a concentration of 2 mg/kg or 1 mg/kg results in the growth inhibition of broilers,and reduces the hepatic GSTs activity of broilers,and the longer time the AFB₁ exposure, the more obvious of the inhibition on the hepatic GSTs activity.（2） the effects of AFB₁ on the the gene expression of eight kinds of hepatic glutathione S-transferase and the protein expression of four kinds of typical hepatic glutathione S-transferase enzyme in broilers: The mRNA levels of eight hepatic GSTs, including GSTA 2,GSTA1, GSTA3, GSTA-CL3, GSTT1, GSTZ1, GSTK1, and GSTO1, were no significant changes after AFB₁ exposure for 7d or 14 d. After AFB₁ exposure for 21 d,the m RNA levels of GSTA1, GSTT1 and GSTO1 were significantly decreased in the 1mg / kg AFB₁ group; the mRNA levels of GSTA1, GSTT1,GSTZ1 and GSTO1 were significantly decreased in the 2mg / kg AFB₁ group;the mRNA levels of GSTA2 was significantly elevated in the 2mg / kg AFB₁ group; the mRNA levels of GSTA3, GSTA-CL3 and GSTK1 were no significant changes in both the 1mg / kg AFB₁ group and the 2mg / kg AFB₁ group.The protein levels of GSTT1 and GSTO1 were significantly decreased after AFB₁ exposure for 21 d,.However, AFB₁ elevated the protein expression of GSTA2. The test showed that diet with 1mg / kg AFB₁ or 2mg / kg AFB₁ 21 d, the mRNA expression of hepatic GSTA1, GSTT1, GSTZ1 and GSTO1 were inhibited by AFB₁ in male broilers, and GSTA1, GSTT1 and GSTO1 may be relatively sensitive to the dose of AFB₁ in feed. Whereas the increase of mRNA and protein expression of GSTA2 in male broilers may be a stress response to AFB₁, and it also indicated that GSTA2 might contribute to the resistance of broilers to AFB₁.