Bromodeoxyuridine mutagenesis in mammalian cells: mutagenesis is independent of the amount of bromouracil in DNA.

Abstract

Studies were undertaken to determine how a line of mutant Syrian hamster melanoma cells (HAB-2E) that displays unlimited growth potential when all of the thymine residues in nuclear DNA are replaced by bromouracil (BrUra) could avoid the deleterious effects of bromodeoxyuridine (BrdUrd) mutagenicity. It was found that BrdUrd could be mutagenic to these cells. However, there was a nonlinear relationship between mutagenicity and the amount of BrUra in the DNA of the HAB-2E cells. With these cells, mutagenicity apparently is determined by the concentration of BrdUrd to which the cells are exposed rather than the amount of BrUra in DNA. These results were obtained with both the induction of ouabain resistance and thioguanine resistance as markers for mutagenesis. The dependence of BrdUrd mutagenicity on BrdUrd concentration was also observed for the parental melanoma cells.

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

“…A disadvantage to this method is the inherent toxicity of nucleoside analogs such as BrdU, FdU, and IdU (Sivakumar et al 2004). Observations in bacteria and mammals indicate that BrdU is both a mutagen and a teratogen Kaufman and Davidson 1978;Lasken and Goodman 1984), able to cause T-C transition mutations . However, up to 400 mg/ml BrdU may be added to a culture of S. cerevisiae before toxic effects are observed (Lengronne et al 2001).…”

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

“…The FBT medium contains BrdUrd, dThd, and FdUrd, an inhibitor of de novo thymidylate biosynthesis (6). Under these culture conditions, the ratio of BrUra to Thy residues in newly synthesized DNA was determined by the ratio of BrdUrd to dThd in the culture medium (13). In this medium, the substitution of BrUra for Thy residues in DNA could be varied at a constant BrdUrd concentration by varying only the dThd concentration.…”

“…Conversely, the level of BrUra substituted for Thy could be held constant while the BrdUrd concentration in the medium was varied, by maintaining a constant ratio of BrdUrd to dThd. This protocol was used to confirm the relationship between BrdUrd concentration and mutagenesis previously described (13) and to provide a basis for comparing pool-dependent mutagenesis with DNA-dependent mutagenesis. In addition, this protocol was used to determine the mechanism by which deoxyguanosine (dGuo) stimulates BrdUrd mutagenesis, a phenomenon previously described (14) but not elucidated.…”

“…Recent studies in mammalian systems suggested that BrdUrd mutagenesis was determined primarily by the concentration of BrdUrd to which the cells were exposed and was independent of the amount of BrUra incorporated into DNA in place of Thy residues (13). 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.…”

“…The thymidine (dThd) analog 5-bromodeoxyuridine (BrdUrd) is an effective mutagen in procaryotic (3,9,16,20,23) and eucaryotic (1,12,13,22) systems. A model for the mutagenic action of this base analog was based on the Watson and Crick scheme of base pairing and the suggestion that rare tautomeric shifts of the bases could change their pairing properties during DNA replication and thereby lead to mutations (9).…”