A novel assay of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity in cultured cells and its use for evaluation of cadmium(II) inhibition of this activity.
8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of oxidative modification of dGTP, thatcan be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to 8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer. To elucidate the involvement of 8-oxo-dGTPases in carcinogenesis, an assay of the 8-oxo-dGTPase activity is required. This paper presents such an assay developed for Chinese hamster ovary (CHO) cells that can be applied to any biological material. It includes: (i) a convenient method for preparing 8-oxo-2'-deoxyguanosine 5'-phosphates; (ii) an HPLC/UV quantification of 8-oxo-dGTP hydrolysis products and (iii) separation of 8-oxo-dGTPase activity from interfering 8-oxo-dGTP phosphatase(s). The 8-oxo-dGTPase activity of CHO cells depends on magnesium, has a pH optimum of 8.5, Km for 8-oxo-dGTP of 9.3 microM, and is inhibited by 8-oxo-dGDP, the product of interfering 8-oxo-dGTP phosphatases. The latter must be removed from the assayed samples by ultrafiltration through 30 kDa cut-off membranes. The method was used to test the inhibition by cadmium ions of the activity of 8-oxo-dGTPase in CHO cells. The cells cultured with 0.3-3 microM cadmium(II) acetate for up to 24 h had their 8-oxo-dGTPase activity suppressed in a Cd(II) concentration-dependent manner, down to 70% of the control value.
Bialkowski
K
K
Laboratory of Comparative Carcinogenesis, National Cancer Institute-FCRDC, Building 538, Room 205E, Frederick, MD 21702, USA. karolb@mail.ncifcrf.gov
Kasprzak
K S
KS
D000818:Animals; D016466:CHO Cells; D002104:Cadmium / Q000494:pharmacology*; D002848:Chromatography, DEAE-Cellulose; D006224:Cricetinae; D045643:DNA Repair Enzymes; D003848:Deoxyguanine Nucleotides / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005069:Evaluation Studies as Topic; D006868:Hydrolysis; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results
D003848:Deoxyguanine Nucleotides; D004791:Enzyme Inhibitors; D002104:Cadmium; D010744:Phosphoric Monoester Hydrolases; C078207:8-oxodGTPase; D045643:DNA Repair Enzymes