Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.
Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.
Trinidad
A C
AC
Biomedical Sciences Division, Lawrence Livermore National Laboratory, California.
Wu
R W
RW
Thompson
L H
LH
Felton
J S
JS
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001564:Benzo(a)pyrene / Q000378:metabolism; D016466:CHO Cells / Q000201:enzymology*; D003001:Cloning, Molecular; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004260:DNA Repair; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays
D011994:Recombinant Proteins; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases