The aflatoxin B1 degrading abilities of two different ruminants were compared in this study. One set of experiments evaluated the aflatoxin B1 degradation ability of different rumen fluid donors (steers vs. goats) as well as the rumen fluid filtration method (cheese cloth filtered vs. 0.45 microm Millipore) in a 2 x 2 factorial arrangement. Additional studies examined aflatoxin B1 degradation by collecting rumen fluid at different times (0, 3, 6, 9 and 12 h) after feeding. Cannulated Holstein steers (740 +/- 10 kg bw) and Korean native goats (26 +/- 3 kg bw) were fed a 60% timothy and 40% commercial diet with free access to water. Rumen fluid from Korean native goats demonstrated higher (p < 0.01) aflatoxin B1 degradability than Holstein steers. However, filtration method had no significant influence on degradability. In addition, aflatoxin degradation did not depend upon rumen fluid collection time after feeding, as no significant differences were observed. Finally, a comparison of two types of diet high in roughage found aflatoxin degradability in goats was higher with timothy hay opposed to rice straw, although individual variation existed. Thus, our findings showed the aflatoxin degradability is comparatively higher in goats compared to steers.
Aflatoxin B1/*chemistry/*metabolism ; Animals ; Body Fluids/*chemistry/metabolism ; Cattle/*physiology ; Goats/*physiology ; Korea ; Male ; Rumen/*metabolism

OBJECTIVETo establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity.

METHODSThe human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test.

RESULTThe HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells.

CONCLUSIONThe established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.

Aflatoxin B1 ; metabolism ; Biotransformation ; Cell Line ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; DNA, Complementary ; chemistry ; Humans ; RNA, Messenger ; analysis

Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization.
Absorption ; Aflatoxin B1 ; metabolism ; toxicity ; Animals ; Chickens ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Multienzyme Complexes ; chemistry ; metabolism ; Rats ; Temperature ; Toxicity Tests

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering