The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.     

Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells

Platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) catalyzes the phosphorolysis of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P) and has a pro-angiogenic effect for which dR-1-P may be responsible. Using a purine nucleoside phosphorylase based assay it was found that TdR incubation did not increase dR-1-P accumulation in colon cancer cell line Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. The assay was linear up to 25,000pmol dR-1-P with complete recovery of dR-1-P from cellular extracts. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a thymidine phosphorylase inhibitor. dR-1-P directly added to cellular extracts disappeared within 5-10min. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells.     

Role of platelet derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1-phosphate

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P). TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. We investigated the role of TP in 5'-deoxy-5-fluorouridine (5'DFUR), 5-fluorouracil (5FU) and trifluorothymidine (TFT) sensitivity. TP had no effect on TFT while it activated 5'DFUR and to a lesser extent 5FU. In order to provide an explanation for this difference in activation of 5'DFUR and 5FU, we studied the role of the 5FU co-substrate, dR-1-P, needed for its activation.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.     

Intracellular Thymidylate Synthase Inhibition by Trifluorothymidine in FM3A Cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5‐Fluorouracil (5FU). TS activity reduced to 9% (0.1 µM TFT) and 58% (1 µM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK^?) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Intracellular thymidylate synthase inhibition by trifluorothymidine in FM3A cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5-Fluorouracil (5FU). TS activity reduced to 9% (0.1 microM TFT) and 58% (1 microM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK-) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Role of Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase in Fluoropyrimidine Sensitivity and Potential Role of Deoxyribose‐1‐Phosphate

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine (TdR) to thymine and deoxyribose‐1‐phosphate (dR‐1‐P). TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. We investigated the role of TP in 5′‐deoxy‐5‐fluorouridine (5′DFUR), 5‐fluorouracil (5FU) and trifluorothymidine (TFT) sensitivity. TP had no effect on TFT while it activated 5′DFUR and to a lesser extent 5FU. In order to provide an explanation for this difference in activation of 5′DFUR and 5FU, we studied the role of the 5FU co‐substrate, dR‐1‐P, needed for its activation.     

Estrogen receptor beta mRNA in colon cancer cells: growth effects of estrogen and genistein

Knowledge regarding the expression of the recently cloned estrogen receptor beta (ERbeta) in colonic mucosa is limited. In this study, we demonstrated that five human colon cancer cell lines, HT29, Colo320, Lovo, SW480, and HCT116, expressed ERbeta mRNA, but lacked ERalpha mRNA. Results from a cell growth assay demonstrated that these colon cancer cells were not influenced by estrogen, while genistein possessed slight growth inhibitory effects on HT29, Colo320 and Lovo cells at 10 microM, at which concentration is stimulated the growth of ERalpha-positive human breast cancer MCF-7 cells. Tamoxifen inhibited the growth of HT29 and Colo320 cells, dose-dependently, as well as MCF-7 cells. A transfected reporter plasmid containing a vitellogenin estrogen response element could be activated by estradiol in Colo320 cells. Taken together with previous reports, these data suggest that ERalpha and ERbeta may have different biological functions in colon cells.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Knockdown of DNA‐binding protein A enhances the chemotherapy sensitivity of colorectal cancer via suppressing the Wnt/β‐catenin/Chk1 pathway

DNA‐binding protein A (dbpA) is reported to be upregulated in many cancers and associated with tumor progress. The present study aimed to investigate the role of dbpA in 5‐fluorouracil (5‐FU)‐resistant and oxaliplatin (L‐OHP)‐resistant colorectal cancer (CRC) cells. We found that 5‐FU and L‐OPH treatment promoted the expression of dbpA. Enhanced dbpA promoted the drug resistance of SW620 cells to 5‐FU and L‐OHP. DbpA knockdown inhibited cell proliferation, induced cell apoptosis, and cell cycle arrested in SW620/5‐FU and SW620/L‐OHP cells. Besides, dbpA short hairpin RNA (shRNA) enhanced the cytotoxicity of 5‐FU and L‐OHP to SW620/5‐FU and SW620/L‐OHP cells. Meanwhile, dbpA shRNA inhibited the activation of the Wnt/β‐catenin pathway that induced by 5‐FU stimulation in SW620/5‐FU cells. Activation of the Wnt/β‐catenin pathway or overexpression of checkpoint kinase 1 (Chk1) abrogated the promoting effect of dbpA downregulation on 5‐FU sensitivity of CRC cells. Importantly, downregulation of dbpA suppressed tumor growth and promoted CRC cells sensitivity to 5‐FU in vivo. Our study indicated that the knockdown of dbpA enhanced the sensitivity of CRC cells to 5‐FU via Wnt/β‐catenin/Chk1 pathway, and DbpA may be a potential therapeutic target to sensitize drug resistance CRC to 5‐FU and L‐OHP.     

Metabolism of pyrimidine bases and nucleosides in Bacillus subtilis.

In Bacillus subtilis, uracil (Ura), uridine (Urd), and deoxyuridine (dUrd) are metabolized through pathways similar to those of enteric bacteria. Ura is probably converted to uridine 5'-monophosphate by uridine 5'-monophosphate pyrophosphorylase. More than 95% of dUrd added to cultures is converted to Ura and deoxyribose-1-phosphate. Although dUrd kinase activity is detectable in vitro, this enzyme does not seem to play an important role in the metabolism of dUrd. The metabolism of cytosine (Cyt), cytidine (Cyd), and deoxycytidine (dCyd) in B. subtilis appears to be different from that in enteric bacteria. Cytosine cannot be used by Ura-requiring mutants as pyrimidine source. dCyd is deaminated by dCyd-Cyd deaminase or phosphorylated to dCyd nucleotides by dCyd kinase. Cyd is deaminated by dCyd-Cyd deaminase of phosphorylated by Cyd kinase. This Cyd kinase activity has never been reported for B. subtilis.     

Selective Protection by Uridine of Growth Inhibition by 5-Fluorouracil (5FU) Mediated by 5FU Incorporation into RNA,But Not the Thymidylate Synthase Mediated Growth Inhibition by 5FU-Leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.     

Selective protection by uridine of growth inhibition by 5-fluorouracil (5FU) mediated by 5FU incorporation into RNA, but not the thymidylate synthase mediated growth inhibition by 5FU-leucovorin

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.     

microRNA‐577 suppresses tumor growth and enhances chemosensitivity in colorectal cancer

In this work, we aimed to determine the expression and biological functions of microRNA (miR)‐577 in colorectal cancer (CRC). The results showed that miR‐577 was downregulated in CRC specimens and cell lines. Restoration of miR‐577 significantly suppressed the proliferation and colony formation and induced a G0/G1 cell cycle arrest in CRC cells. 5‐Fluorouracil (5‐FU)‐resistant SW480 cells (SW480/5‐FU) were found to have elevated levels of miR‐577. Ectopic expression of miR‐577 enhanced 5‐FU sensitivity in SW480/5‐FU cells. Heat shock protein 27 (HSP27) was identified as a target gene of miR‐577. Enforced expression of HSP27 reversed the effects of miR‐577 on CRC cell growth and 5‐FU sensitivity. Xenograft tumors derived from miR‐577‐overexpressing SW480 cells exhibited significantly slower growth than control tumors. In conclusion, our results support that miR‐577 acts as a tumor suppressor in CRC likely through targeting HSP27. Therefore, miR‐577 may have therapeutic potential in the treatment of CRC.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

Radiosensitization by Thymidine Phosphorylase Inhibitor in Thymidine Phosphorylase Negative and Overexpressing Bladder Cancer Cell Lines

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p <.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ?H2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.     

Deoxyribose protects against rapamycin-induced cytotoxicity in colorectal cancer cells in vitro

Thymidine phosphorylase (TPase) is also known as the platelet-derived endothelial cell growth factor (PD-ECGF) and plays a role in angiogenesis. Deoxyribose (dR; a downstream TPase-product) addition to endothelial cells may stimulate FAK and p70/S6k signaling, which can be inhibited by rapamycin. Rapamycin is a specific mammalian target of the rapamycin (mTOR) inhibitor, a kinase that lies directly upstream of p70/S6k. This suggests a role for TPase in the mTOR/p70/S6k pathway. In order to study this in more detail, we exposed cells with and without TPase expression to dR and rapamycin and determined the effect on cell growth. We observed protection in cytotoxicity in Colo320 cells, but not Colo320 TP1 cells. This was in part mediated by activation of p70/S6k and inhibition of autophagy. Further studies are recommended to elucidate the mechanism behind the protective effect of dR.     

Optimized blood sampling with cytidine deaminase inhibitor for improved analysis of capecitabine metabolites

The 5FU prodrug capecitabine undergoes a 3-step enzymatic conversion, including the conversion of 5'DFRC into 5'DFUR by cytidine deaminase (CDA). The presence of CDA activity in blood led us to analyze the possible ex vivo conversion of 5'DFCR into 5'DFUR in blood samples. We thus examined the impact of the addition of a CDA inhibitor (tetrahydrouridine (THU) 1 microM final) in blood. Blood samples from 3 healthy volunteers were taken on tubes containing or not THU. Blood was spiked with 5'DFCR (20 microM final) (T0) and was maintained at room temperature for 2 h. Plasma concentrations of 5'DFRC and 5'DFUR were analyzed with an optimized HPLC assay. In the absence of THU, 5'DFUR was detectable as early as T0. The percent of 5'DFUR produced relative to 5'DFCR increased over time, up to 7.7 % at 2h. In contrast, the presence of THU totally prevents the formation of 5'DFUR. The impact of THU for preventing the conversion of 5'DFCR was confirmed by the analysis of blood samples from 2 capecitabine-treated patients. Addition of THU in the sampling-tube before the introduction of blood is thus strongly recommended in order to guarantee accurate conditions for reliable measurement of capecitabine metabolites in plasma, and thus faithful pharmacokinetic data.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.