A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.

Effects of 5-hydroxymethyluracil and 3-aminobenzamide on the repair and toxicity of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells.

5-Hydroxymethyl-2'-deoxyuridine (HmdUrd), a cytotoxic analogue of thymidine, has been proposed for use as an anticancer agent. HmdUrd is incorporated into DNA and then removed at a rate of 30-40% per day. The removal of HmdUrd from DNA has been attributed to the action of 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). We demonstrated the release of [3H]HmUra into the growth medium of V79 Chinese hamster cells that had incorporated [3H]HmdUrd into their DNA. The amount of [3H]HmUra recovered from the growth medium was equal to the amount of [3H]HmdUrd lost from DNA. These experiments confirmed that the initial step of repair of HmUra in DNA was mediated by DNA glycosylase activity. A combination of HmUra and HmdUrd resulted in increased uptake of HmdUrd by cells and increased cytotoxicity. The increased incorporation of HmdUrd into DNA was not due to inhibition of repair. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, was cytotoxic to cells which incorporated and repaired HmdUrd. The extent of toxicity was directly related to the number of HmUra residues in DNA. HeLa cells, known to be resistant to the toxic effects of HmdUrd, do not incorporate HmdUrd into their DNA. HeLa cells were resistant to the toxic effects of 3-aminobenzamide, confirming that the absence of HmdUrd in their DNA was not due to an accelerated rate of repair. These experiments indicate that the potential therapeutic antineoplastic properties of HmdUrd may be enhanced by using HmUra to increase the incorporation of HmdUrd into DNA and 3-aminobenzamide to interfere with repair of HmUra in DNA.

Mutagenicity of 5-hydroxymethyl-2'-deoxyuridine to Chinese hamster cells.

5-Hydroxymethyluracil (HmUra) is formed from thymine in DNA through the action of ionizing radiation or reactive oxygen species generated by activated leukocytes. HmUra is removed from DNA by a specific DNA glycosylase, suggesting that it is also formed from endogenously generated reactive oxygen species and that its formation in DNA is potentially deleterious. To determine whether HmUra residues in DNA are mutagenic, hamster V79 cells were grown in the presence of 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) which is incorporated into DNA, and mutagenicity at the ouabain- and thioguanine-resistant loci was determined. Levels of substitution ranged from 1/500 to 1/5,000 HmUra residues/thymine residues. There was slight mutagenicity at the thioguanine-resistant locus but none at the ouabain-resistant locus. The mutagenicity of HmdUrd, expressed as a function of HmUra substitution in DNA, was 1/30,000 in the hypoxanthine-guanine-phosphoribosyltransferase target gene. This low frequency indicates that the oxidation of thymine to HmUra in a preexisting AT base pair does not contribute significantly to the mutagenicity of ionizing radiation, because the yield of HmUra formed in DNA at mutagenic doses of radiation is too low. To determine whether repair of HmUra might be inhibited by ionizing radiation, cells were grown in medium containing HmdUrd and exposed to as much as 5 Gy of gamma-irradiation, and the removal of HmUra from DNA was measured. No inhibition of repair was noted. Preirradiation of cells neither accelerated the rate of repair nor raised the level of HmUra-DNA glycosylase activity, indicating that repair of HmUra was not induced by this type of oxidative stress. Although the mutagenicity of HmUra residues in DNA is low, even a rare mutation might be sufficiently deleterious to higher organisms to promote the development of HmUra-DNA glycosylase activity.

Toxicity of 3-aminobenzamide to Chinese hamster cells containing 5-hydroxymethyluracil in their DNA.

V79 cells incorporated 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) into their DNA linearly over a wide range of concentrations and time. Cells grew normally when 0.03% of thymidine residues were replaced with HmdUrd. At this level of substitution, 5-hydroxymethyluracil (HmUra) was removed from DNA at a rate of 30-40%/24 h. Concentrations of HmdUrd in the growth medium which produced higher levels of substitution reduced survival and caused cells to delay their transit through S phase. However, the treatment of HmdUrd-containing cells with 3-aminobenzamide caused extensive cell death. At levels of HmdUrd substitution compatible with near 90% survival, the addition of 3-aminobenzamide, an inhibitor of poly (adenosine diphosphoribose) synthesis, killed over 90% of the cells. This toxicity was not due to inhibition of the removal of HmUra from DNA. Cells killed by this combination of agents arrested in the G2 phase of the cell cycle. We conclude that the toxicity of HmdUrd resulted primarily from the repair of the HmUra residue in DNA and not from any intrinsic toxicity of the HmUra residue itself. We also conclude that the cytotoxicity of 3-aminobenzamide resulted from interference with the completion of DNA repair following base (HmUra) excision. Since HmUra is also formed in DNA through the action of ionizing radiation, it may be among the components of radiation-induced DNA damage which sensitizes cells to 3-aminobenzamide.

Radiation-like modification of bases in DNA exposed to tumor promoter-activated polymorphonuclear leukocytes.

Oxygen species generated by human polymorphonuclear leukocytes (PMNs) activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) caused the formation of 5-hydroxymethyl-2'-deoxyuridine (HMdUrd), and (+) and (-) diastereoisomers of cis-thymidine glycol (dTG) in DNA that was exposed to them. There were 9 HMdUrds and 31 dTGs formed per 1 X 10(6) thymidine residues. When Fe(II)/ethylenediaminetetraacetic acid was added to TPA-activated PMNs at 0, 10, 15, and 20 min after TPA, HMdUrd formation increased 5-, 13-, 30-, and 35-fold. Although dTG was initially formed in larger amounts than HMdUrd, it eventually decreased but was still 5-, 6-, 5.5-, and 3-5-fold, respectively, higher than in the absence of iron. From 65 to 1800 times more HMdUrd was formed in DNA when autologous plasma was present during incubation of DNA with TPA-activated PMNs than in its absence. The levels of dTG also varied from about the same as HMdUrd to the nondetectable. Reconstituted human serum transferrin used instead of plasma or Fe(II) also supported the formation of HMdUrd and dTG. When DNA was treated with Fe(II)-reduced H2O2 in the absence of PMNs and TPA, both derivatives were formed. However, the same treatment of marker dTG of dTG-containing polydeoxyadenylic-thymidylic acid caused the decomposition of dTG. Thus, the reduction of hydrogen peroxide by Fe(II) complexed to either ethylenediaminetetraacetic acid or amino acids amy be responsible for the formation of HMdUrd and dTG and for subsequent decomposition of dTG in DNA exposed to the TPA-activated PMNs.

Normal endonuclease activities for damaged DNA during hepatocarcinogenesis.

Two endonuclease activities in rat liver for damaged DNA were assayed. Double-stranded, covalently closed DNA from phage PM2 was damaged by either ultraviolet irradiation or by heating at acid pH, and used as substrate for endonucleases specific for ultraviolet DNA damage and for DNA apurinic sites, respectively. The levels of both enzyme activities in livers of normal rats were compared to levels in livers of rats fed N-2-acetylaminofluorene. At critical stages of the carcinogenic regimen levels of both endonuclease activities were normal. This, together with other data, suggests that depression of excision-repair of DNA damage does not take place during experimental carcinogenesis.

Identification of radiation-induced thymine derivatives in DNA.

A methodology for the separation of radiation-induced thymine derivatives in DNA using high pressure liquid chromatography is presented. DNA was subjected to enzymatic hydrolysis yielding 2'-deoxyribonucleosides and the hydrolysate cochromatographed with marker compounds. Confirmation of the presence of derivatives was accomplished by chromatography on Sephadex LH-20 and microderivatization. The method separates free bases from nucleosides allowing for identification of spontaneously released bases or those released through the action of repair enzymes. The results indicate that most of the thymine derivatives formed in irradiated cellular DNA were the same as those found in DNA irradiated in solution. However, the major cellular derivative was not present in the latter. This derivative was identified as 5-hydroxymethyl-2'-deoxyuridine (HMdU). HMdU has previously been shown to be cytotoxic to cells in culture and caused diarrhea and bone marrow failure when administered to mice. Thus, the presence of this radiation-induced thymine derivative in cellular DNA correlates with the known effects of ionizing radiation on cells and animals.

The repairability of oxidative free radical mediated damage to DNA: a review.

Many DNA repair enzyme activities are present in both prokaryotic and eukaryotic organisms. Among these are DNA exo- and endonucleases and DNA glycosylases which remove oxidatively damaged portions of the DNA molecule, thereby initiating excision-repair. The existence of these enzymes may be taken as evidence that cellular DNA is continuously subject to endogenous oxidative stress. Many of the lesions introduced by ionizing and ultraviolet radiation are identical to those introduced into DNA by reactive oxygen species generated by activated white cells, and are substrates for the repair enzymes. The chemical nature of the lesions, their biologic effects, and the mechanism of their repairability are described.

5-Hydroxymethyluracil-DNA glycosylase activity may be a differentiated mammalian function.

To determine the prevalence of the repair enzyme HMU-DNA glycosylase we assayed its activity in whole cell extracts of several bacterial species, the eukaryotic yeast Saccharomyces cerevisiae, mammalian cell lines and murine tissue. Enzyme activity was constitutively present in murine, hamster and human cell lines. It was not inducible by exposing cells to oxidative stress from ionizing radiation or by incubating cells with the 2'-deoxynucleoside of HMU, HMdU. In murine tissue, enzyme activity was highest in brain and thymus. HMU-DNA glycosylase activity was not detectable in bacteria or yeast nor could activity be detected after exposure of cells to H2O2. These results suggest that, in contrast to other DNA-repair enzymes, HMU-DNA glycosylase is a differentiated function limited to higher eukaryotic organisms.

Genetic effects of 5-hydroxymethyl-2'-deoxyuridine, a product of ionizing radiation.

Ionizing radiation causes formation of heterogeneous types of damage to DNA. Among those, 5-hydroxymethyl-2'-deoxyuridine (HMdU) was identified as a major thymidine derivative in gamma-irradiated HeLa cells [G.W. Teebor, K. Frenkel and M.S. Goldstein (1984) Proc. Natl. Acad. Sci. (U.S.A.), 81, 318-321]. We report here that HMdU is a strong inducer of lambda prophage in Escherichia coli WP2s(lambda) and is highy mutagenic in Salmonella typhimurium. HMdU causes his+ revertants in strains TA100, which reverts predominantly by base-pair substitution at G-C sites, and TA97, which reverts mainly by frameshift mutation at G-C sites. It does not cause reversion in TA98, another frameshift-sensitive strain, nor in strains TA1535 and TA1537. Of those tested, only the last two strains do not contain pkM101, a plasmid which enhances mutagenic effects of ionizing radiation. HMdU also causes reversion in strains TA102 and TA104, which detect oxidative damage and can revert by base-pair substitution at A-T base pairs at the hisG428 site. We show that HMdU can be incorporated into DNA of TA100 and that, in addition to causing point mutations, it causes suppressor mutations as well. The ability of HMdU to induce lambda prophage and its strong mutagenicity in Salmonella typhimurium provide evidence that the presence of HMdU in DNA is biologically significant and may play a major role in the genetic consequences of ionizing radiation and other types of oxidative damage.

Site directed substitution of 5-hydroxymethyluracil for thymine in replicating phi X-174am3 DNA via synthesis of 5-hydroxymethyl-2'-deoxyuridine-5'-triphosphate.

5-hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of Thy. It is removed from DNA by HmUra-DNA glycosylase. To determine whether the replacement of Thy by HmUra is mutagenic, which might explain the repairability of HmUra, a HmUra residue was substituted for Thy in a target (amber) codon by in vitro extension of an oligonucleotide primer annealed to phi X-174am3 virion DNA. This was accomplished by synthesizing HmdUTP and using DNA polymerase to effect primer extension. E. coli spheroplasts were transfected with the HmUra-containing DNA and the yield of revertant phage determined following replication in the bacterial host. Since E. coli do not express HmUra-DNA glycosylase activity, mutagenesis could be assessed in the absence of repair. chi 2c analysis showed that replacing Thy with HmUra did not result in an increase in revertant phage. These data indicate that the oxidation of Thy to HmUra in cellular DNA probably does not result in substantial mutagenesis.

Detection of different types of damage in alkylated DNA by means of human corrective endonuclease (correndonuclease).

Corrective endonuclease (correndunclease) activity of HeLa cells was assayed with alkylated DNA. Double-stranded, covalently closed DNA from phage PM II was treated with methyl methanesulfonate, N-methyl-N-nitrosourea, beta-propiolactone, or diepoxybutane to introduce alkylated bases and alkali-labile sites into the DNA. The damaged DNA was incubated with an extract of HeLa cells that catalyzes the formation of breaks at apurinic sites in double-stranded DNA. Methylated DNA was broken at every alkali-labile site by the HeLa correndonuclease, which indicated that these sites are similar to the apurinic sites produced by heating at acid pH. DNA alkylated with beta-propiolactone or diepoxybutane containing the same number of alkali-labile sites was broken to a far lesser extent. This indicates the presence of a second type of alkali-labile damage that is correndonuclease-insensitive.

Identification of the cis-thymine glycol moiety in chemically oxidized and gamma-irradiated deoxyribonucleic acid by high-pressure liquid chromatography analysis.

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by chemical oxidants and ionizing radiation. We describe the separation of thymine glycol, 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol), thymine, and thymidine by high-pressure liquid chromatography (HPLC). Enzymatic hydrolysates of chemically oxidized or gamma-irradiated single-stranded DNA were cochromatographed with 14C-containing marker compounds. In chemically oxidized DNA, thymidine glycol was the major derivative formed. In addition, there were four rapidly eluting thymine-derived components. In irradiated DNA, thymidine glycol constituted about 5% of the modified thymines, and the rapidly eluting fractions were proportionately increased. DNA isolated from gamma-irradiated and nonirradiated HeLa cells grown in the presence of [3H]thymidine was subjected to enzymatic hydrolysis and HPLC analysis. In control DNA, 0.3% of the thymines were modified. Thirty-six kilorads of gamma radiation caused a 30% increase in thymine damage. Thus, most of the base damage was due to internal beta radiation from incorporated [3H]thymidine. The chromatographic patterns of irradiated and nonirradiated samples were qualitatively the same, but the yields of some products increased 2-fold, while others remained unchanged. A comparison of the HPLC profiles of hydrolysates of in vitro oxidized and irradiated DNA with those of the cellular DNA revealed one fast eluting peak to be absent in cellular DNA, suggesting that it was formed only in single-stranded DNA. In cellular DNA, the major modified thymine was a more hydrophobic derivative not formed by in vitro radiation nor chemical oxidation. As in in vitro irradiated DNA, thymidine glycol constituted 5% of the modified thymines. The presence of cis-thymidine glycol in hydrolysates was confirmed by chromatography on Sephadex LH-20 using water and borate as eluants.

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.