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, that can be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTPases) 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-dQTPase activity of CHO cells depends on magnesium, has a pH optimum of 8.5, K-m for 8-oxo-dGTP of 9.3 mu M, 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 mu M 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. [References: 28]

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