The effect of UDP-glucuronosyltransferase 1A1 expression on the mutagenicity and metabolism of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in CHO cells.
UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies, UGT1A1 has been implicated in the detoxification of certain food-borne carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDP-glucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neo gene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of 11 clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52-kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining four clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10(4)-10(5)-fold higher relative to the UV5P3 parental cells. One clone (#14) had a 10-fold higher increase in expression at 1.47 x 10(5) over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D50 values, the cytotoxic effect of PhIP was decreased approximately 350-fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition, no significant increase in mutation frequency was observed in the transfected cells. These results clearly indicate that UGT1A1 plays a critical role in PhIP biotransformation, providing protection against PhIP-mediated cytotoxicity and mutagenicity.
Malfatti
Michael A
MA
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, P.O. Box 808, L-452, Livermore, CA 94551-9900, USA.
Wu
Rebekah W
RW
Felton
James S
JS
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002273:Carcinogens / Q000378:metabolism / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism / Q000633:toxicity*; D016296:Mutagenesis; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002273:Carcinogens; D018076:DNA, Complementary; D007093:Imidazoles; D009153:Mutagens; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D014453:Glucuronosyltransferase