Porter D W, Yakushiji H, Nakabeppu Y, Sekiguchi M, Fivash M J, Kasprzak K S
Laboratory of Comparative Carcinogenesis, National Cancer Institute-FCRDC, Frederick, MD 21702, USA.
Carcinogenesis. 1997 Sep;18(9):1785-91. doi: 10.1093/carcin/18.9.1785.
The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in cultured cells, has been associated with generation of the promutagenic lesion 8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects. One possible source of this base may be 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool, from which it is incorporated into DNA. To promote such incorporation, the metals would have to inhibit specific cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool. The present study was designed to test such inhibition in vitro on 8-oxo-dGTPases from two different species, the human MTH1 protein and Escherichia coli MutT protein. In the presence of Mg(II), the natural activator of 8-oxo-dGTPases, all four metals were found to inhibit both enzymes. For MTH1, the IC50 values (+/- SE; n = 3-4) were 17 +/- 2 microM for Cu(II), 30 +/- 8 microM for Cd(II), 376 +/- 71 microM for Co(II) and 801 +/- 97 microM for Ni(II). For MutT, they were 60 +/- 6 microM for Cd(II), 102 +/- 8 microM for Cu(II), 1461 +/- 96 microM for Ni(II) and 8788 +/- 1003 microM for Co(II). Thus, Cu(II) and Cd(II) emerged as much stronger inhibitors than Ni(II) and Co(II), and MTH1 appeared to be generally more sensitive to metal inhibition than MutT. Interestingly, in the absence of Mg(II), the activity of the enzymes could be restored by Co(II) to 73% of that with Mg(II) alone for MutT, and 34% for MTH1, the other metals being much less or non-effective. The difference in sensitivity to metal inhibition between the two enzymes may reflect the differences in the amino acid ligands, especially the cysteine ligand, outside their evolutionarily conserved Mg(II)-binding active sites, which might indicate predominantly non-competitive or uncompetitive mechanism of the inhibition. The overall results suggest that inhibition of 8-oxo-dGTPases may be involved in the mechanisms of induction of the 8-oxoguanine lesion in DNA by the metal ions studied, especially the non-redox-active Cd(II) cation.
镍（II）、钴（II）和铜（II）对动物的毒性，以及镉（II）对培养细胞的毒性，与DNA中促诱变损伤8-氧代-7,8-二氢鸟嘌呤（8-氧代鸟嘌呤）的产生等效应有关。这种碱基的一个可能来源可能是8-氧代-7,8-二氢-2'-脱氧鸟苷-5'-三磷酸（8-氧代-dGTP），它是核苷酸池氧化损伤的产物，并从该池中掺入DNA。为了促进这种掺入，金属必须抑制特定的细胞8-氧代-dGTP酶，这些酶可从核苷酸池中消除8-氧代-dGTP。本研究旨在体外测试两种不同物种的8-氧代-dGTP酶，即人类MTH1蛋白和大肠杆菌MutT蛋白，对这种抑制作用的敏感性。在8-氧代-dGTP酶的天然激活剂镁（II）存在的情况下，发现所有四种金属都能抑制这两种酶。对于MTH1，铜（II）的IC50值（±标准误；n = 3 - 4）为17 ± 2 μM，镉（II）为30 ± 8 μM，钴（II）为376 ± 71 μM，镍（II）为801 ± 97 μM。对于MutT，镉（II）的IC5值为60 ± 6 μM，铜（II）为102 ± 8 μM，镍（II）为1461 ± 96 μM，钴（II）为8788 ± 1003 μM。因此，铜（II）和镉（II）比镍（II）和钴（II）表现出更强的抑制作用，并且MTH1似乎总体上比MutT对金属抑制更敏感。有趣的是，在没有镁（II）的情况下，钴（II）可将MutT酶的活性恢复到仅存在镁（II）时的73%，将MTH1酶的活性恢复到34%，而其他金属的恢复作用要小得多或没有效果。两种酶对金属抑制的敏感性差异可能反映了它们在进化上保守的镁（II）结合活性位点之外的氨基酸配体，特别是半胱氨酸配体的差异，这可能表明主要是抑制的非竞争性或反竞争性机制。总体结果表明，8-氧代-dGTP酶的抑制作用可能参与了所研究的金属离子诱导DNA中8-氧代鸟嘌呤损伤的机制，尤其是非氧化还原活性的镉（II）阳离子。
