Genetically modified Chinese hamster ovary cells for investigating sulfotransferase-mediated cytotoxicity and mutation by 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine.
To test the hypothesis that the sulfotransferase gene plays a role in the phase II bioactivation of PhIP, a heterocyclic amine found in cooked meats, we transfected the UV5P3 cell line with cDNA plasmids of human aryl sulfotransferases (HAST1 and HAST3). UV5P3 is a nucleotide excision repair-deficient and P4501A2-expressing CHO cell line that we have previously developed. Functionally transformed clones were identified by the differential cytotoxicity (DC) assay that used PhIP as the cytotoxic agent. Two clones designated 5P3H1 and 5P3H3, expressing HAST1 and HAST3, respectively, were chosen for further characterization. Correct fragment sizes of the sulfotransferase cDNAs were identified in both cell lines by polymerase chain reaction. Immunoblot analysis confirmed the expression of the sulfotransferase proteins. The addition of the sulfotransferase inhibitor DCNP decreased the cytotoxic effects of PhIP in a dose-dependent manner. The increase in cell growth was 6. 5-fold for 5P3H1 and 2.4-fold for 5P3H3, relative to values obtained without DCNP. Based on D(50) values, the dose that reduced the survival to 50% relative to untreated controls, the cytotoxic effect of PhIP was increased threefold for 5P3H1 and 1.87-fold for 5P3H3 cell lines over the parental UV5P3 line. There was also a small increase in the mutation response at the aprt locus. These newly established 5P3H1 and 5P3H3 sulfotransferase-expressing cells provide valuable mechanistic information of the bioactivation of PhIP and related compounds. Environ. Mol. Mutagen. 35:57-65, 2000. Published 2000 Wiley-Liss, Inc.
Wu
R W
RW
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-0808, USA. wu2@llnl.gv
Panteleakos
F N
FN
Kadkhodayan
S
S
Bolton-Grob
R
R
McManus
M E
ME
Felton
J S
JS
D000818:Animals; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000633:toxicity*; D007527:Isoenzymes / Q000378:metabolism*; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007527:Isoenzymes; D009153:Mutagens; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D015239:Arylsulfotransferase