Heterologous co-expression of human cytochrome P450 1A2 and polymorphic forms of N-acetyltransferase 2 for studies on aromatic amines in V79 Chinese hamster cells.
V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2-encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promoter-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and N-sulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4 pmol resorufin/min/mg total protein. The N-acetylation of sulphametazine ranged from 0.07 to 1.7 nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolism dependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells co-expressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.
Scheuenpflug
Jürgen
J
GenPharmTox BioTech AG, Research and Development, Fraunhoferstrasse 9, 82152 Planegg/Martinsried, Germany.
Krebsfänger
Niels
N
Doehmer
Johannes
J
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005449:Fluorenes / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D005818:Genetic Engineering; D005822:Genetic Vectors; D008099:Liver / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009152:Mutagenicity Tests / Q000379:methods; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods
D017931:DNA Primers; D018076:DNA, Complementary; D005449:Fluorenes; C012177:2-aminofluorene; D019388:Cytochrome P-450 CYP1A2; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human