Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

Abstract

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.Recent studies from our laboratory have suggested that mutagenesis by 5-bromodeoxyuridine (BrdUrd) in mammalian cells involves perturbations of deoxyribonucleotide metabolism. These studies have demonstrated that (i) the extent of mutagenesis is determined by the concentration of BrdUrd to which the cells are exposed rather than by the amount of BrdUrd incorporated into nuclear deoxyribonucleic acid (DNA); (ii) deoxycytidine (dCyd) can suppress BrdUrd mutagenesis without decreasing the amount of BrdUrd incorporated into DNA; and (iii) thymidine (dThd), deoxyguanosine, and deoxyadenosine can stimulate BrdUrd mutagenesis without increasing the amount of BrdUrd incorporated into DNA (5, 9, 10). The triphosphates of BrdUrd and of the three deoxyribonucleosides which stimulate BrdUrd mutagenesis are all known to inhibit the ribonucleotide reductase-catalyzed reduction of cytidine diphosphate to deoxycytidine diphosphate (13,15,18). Thus, agents which inhibit synthesis of dCyd nucleotides stimulate BrdUrd mutagenesis, whereas dCyd itself suppresses mutagenesis. These results suggest that the perturbation of dCyd metabolism plays a key role in BrdUrd mutagenesis in mammalian cells.The experiments in this study were designed to assess directly the relationship between BrdUrd mutagenesis and nucleotide metabolism by monitoring the changes in deoxyribonucleoside triphosphate (dNTP) pools during BrdUrd mutagenesis and dCyd suppression of mutagenesis. The deoxycytidine triphosphate (dCTP), bromodeoxyuridine triphosphate (BrdUTP) and thymidine triphosphate (TTP) pools were measured at times aft...

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

“…The increased mutation frequency induced by increasing BrdUrd concentration correlated with an increase in the ratio of BrdUTP to dCTP (Table 1). This increase was caused by an increase in the size of the BrdUTP pools with increasing BrdUrd concentration, as well as a decrease in the dCTP pool, confirming previous findings (2).…”

“…This and other studies (2,7,8,14) provided evidence that BrdUrd mutagenesis in mammalian cells was driven by an increase in the intracellular ratio of BrdUTP to dCTP and occurred only during incorporation of BrdUTP into DNA and that mutations due to replication of BrUra residues in DNA did not occur. (We refer to this mutagenesis as "pool-dependent'" because it is dependent upon the BrdUTP pool levels and not the level of BrUra incorporated into DNA.…”

Presence of Dichlorodiphenyltrichloroethane (DDT) in Croatia and Evaluation of Its Genotoxicity

“…Recent studies revealed the nucleotide pool imbalance can have severe consequences on DNA metabolism and it is critical in SCE formation. The modulation of SCE by DNA precursors raises the possibility that DNA changes are responsible for the induction of SCE and mutations in mammalian cells (Popescu, 1999;Ashman & Davidson, 1981). While increased levels of CA have been associated with increased cancer risk (Hagmar et al, 1994(Hagmar et al, , 1998, a similar conclusion has not been reached for SCE or MN.…”

Sequence-Directed Base Mispairing in Human Oncogenes

“…The first step in mutagenesis by dT (or by the dT analog 5-bromodeoxyuridine) apparently is the inhibition of the ribonucleotide reductase-catalyzed reduction of CDP to dCDP, leading to an increase in the intracellular dTTP/dCTP ratio. Subsequently, dTTP, now the nucleotide in excess relative to dCTP, mispairs with template guanine, leading to a GC3AT transition at the next round of replication (2,14,22). We have shown that such mutagenesis by dT (or 5-bromodeoxyuridine) exhibits strong sequence specificity in that it occurs preferentially at the 3Ј G of runs of two or more adjacent G residues, resulting in GC3AT transitions at this position in the multiple guanine run (11,16,17,33).…”