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BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU's effects have not been extensively studied yet. Therefore, we investigated EdU's potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were observed in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concentrations (similar to manufacturers suggested concentration; >5-10 μM) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with additional precautions.

Only a small portion of human immunodeficiency virus type 1 (HIV-1) particles entering the host cell results in productive infection, emphasizing the importance of identifying the functional virus population. Because integration of viral DNA (vDNA) is required for productive infection, efficient vDNA detection is crucial. Here, we use click chemistry to label viruses with integrase coupled to eGFP (HIV IN-eGFP ) and visualize vDNA. Because click labeling with 5-ethynyl-2'-deoxyuridine is hampered by intense background staining of the host nucleus, we opted for developing HIV-1 reverse transcriptase (RT)-specific 2'-deoxynucleoside analogs that contain a clickable triple bond. We synthesized seven propargylated 2'-deoxynucleosides and tested them for lack of cytotoxicity and viral replication inhibition, RT-specific primer extension and incorporation kinetics in vitro , and the capacity to stain HIV-1 DNA. The triphosphate of analog A5 was specifically incorporated by HIV-1 RT, but no vDNA staining was detected during infection. Analog A3 was incorporated in vitro by HIV-1 RT and human DNA polymerase γ and did enable specific HIV-1 DNA labeling. Additionally, A3 supported mitochondria-specific DNA labeling, in line with the in vitro findings. After obtaining proof-of-principle of RT-specific DNA labeling reported here, further chemical refinement is necessary to develop even more efficient HIV-1 DNA labels without background staining of the nucleus or mitochondria., (© 2019 De Wit et al.)

Understanding the molecular dynamics of DNA replication in vivo has been a formidable challenge requiring the development of advanced technologies. Over the past 50 years or so, studies involving DNA autoradiography in bacterial cells have led to sophisticated DNA tract analyses in human cells to characterize replication dynamics at the single-molecule level. Our own lab has used DNA fiber analysis to characterize replication in helicase-deficient human cells. This work led us to propose a model in which the human DNA helicase RECQ1 acts as a governor of the single-stranded DNA binding protein RPA and regulates its bioavailability for DNA synthesis. We have also used the DNA fiber approach to investigate the interactive role of DDX11 helicase with a replication fork protection protein (Timeless) in human cells when they are under pharmacologically induced stress. In this methods chapter, we present a step-by-step protocol for the single-molecule DNA fiber assay. We describe experimental designs to study replication stress and staining patterns from pulse-chase labeling experiments to address the dynamics of replication forks in stressed cells.

During differentiation, neurons progressively restrict their fate repressing the expression of specific genes. Here we describe the involvement in such developmental steps of the methyl-CpG binding protein 2 (MeCP2), an epigenetic factor that participates to chromatin folding and transcriptional regulation. We previously reported that, due to transcriptional impairments, the maturation of Mecp2 null neurons is delayed. To evaluate whether this could stem from altered progenitors proliferation and differentiation, we investigated whether lack of Mecp2 affects these features both in vitro and in vivo. We show that in Mecp2 null embryonic cortexes the expression of genes defining the identity of proliferating neuroprogenitors is enriched and that their permanence in the G1 phase is prolonged. Moreover, the number of cells transitioning from a stage of maturation to a more mature one is increased in Mecp2 null embryonic cortices, in line with the central role of G1 for cell identity refinement. We thus suggest that, possibly due to the lack of proper transcriptional control normally exerted by Mecp2, fate refinement is impaired in developing null cells. We propose that the maturation delay affecting the developing Mecp2 null cortex originates, at least in part, from deranged mechanisms of cell fate refinement.

Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare 'Start' variety using pulse 5-ethynyl-2'-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare 'Start' variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than γ ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes., Competing Interests: The authors declare no conflict of interest.

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2'-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.

An aptamer specifically binding the interleukin-6 receptor and intrinsically comprising multiple units of the nucleoside analogue 5-fluoro-2'-deoxyuridine can exert a cytostatic effect direcly on certain cells presenting the receptor. Thus the modified aptamer fulfils the requirements for active drug targeting in an unprecedented manner. It can easily be synthesized in a single enzymatic step and it binds to a cell surface receptor that is conveyed into the lysosome. Upon degradation of the aptamer by intracellular nucleases the active drug is released within the targeted cells exclusively. In this way the aptamer acts as a prodrug meeting two major prerequisites of a drug delivery system: specific cell targeting and the controlled release of the drug triggered by an endogenous stimulus., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Therapeutic small interfering RNAs (siRNAs) are composed of chemically modified nucleotides, which enhance RNA stability and increase affinity in Watson-Crick base pairing. However, the precise fate of such modified nucleotides once the siRNA is degraded within the cell is unknown. Previously, we demonstrated that deoxythymidine release from degraded siRNAs reversed the cytotoxicity of thymidylate synthase (TS)-targeted siRNAs and other TS inhibitor compounds. We hypothesized that siRNAs could be designed with specific nucleoside analogues that, once released, would enhance siRNA cytotoxicity. TS-targeted siRNAs were designed that contained 5-fluoro-2'-deoxyuridine (FdU) moieties at various locations within the siRNA. After transfection, these siRNAs suppressed TS protein and messenger RNA expression with different efficiencies depending on the location of the FdU modification. FdU was rapidly released from the siRNA as evidenced by formation of the covalent inhibitory ternary complex formed between TS protein and the FdU metabolite, FdUMP. These modified siRNAs exhibited 10-100-fold greater cytotoxicity and induced multiple DNA damage repair and apoptotic pathways when compared with control siRNAs. The strategy of designing siRNA molecules that incorporate cytotoxic nucleosides represents a potentially novel drug development approach for the treatment of cancer and other human diseases.

Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifaceted protein with an essential role in the base excision repair (BER) pathway. Its implication in tumor development, progression, and resistance has been confirmed in multiple cancers, making it a viable target for intensive investigation. In this work, we designed and synthesized different classes of small-molecule inhibitors of the catalytic endonuclease function of APE1 that contain a 3-carbamoylbenzoic acid scaffold. Further structural modifications were made with the aim of increasing the activity and cytotoxicity of these inhibitors. Several of our compounds were shown to inhibit the catalytic endonuclease function of APE1 with potencies in the low-micromolar range in vitro, and therefore represent novel classes of APE1 inhibitors worthy of further development., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [(3)H]thymidine or 5-bromo-2'-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via "click" chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2'-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent evidence has shown that the gemcitabine metabolite, dFdU, is pharmacologically active. Though less potent, dFdU has a longer half-life and could potentiate or antagonize the activity of gemcitabine. Hence, studies were undertaken to evaluate the combined effects. Following chemical synthesis, an improved purification procedure for dFdU was developed (80 % yield; >99 % purity). Zebrafish phenotype-based embryo screens revealed no acute toxicity after gemcitabine or dFdU treatment. Only gemcitabine affected zebrafish development in a dose-dependent manner. Synergy or antagonism for the combination was not observed. Antitumor effects for dFdU were dose dependent. Antagonism was tumor cell-line dependent and did not depend on formation of the intracellular active metabolite of gemcitabine, suggesting that the drug-metabolite interaction occurs later. These studies highlight a platform for testing the pharmacologic activity for anticancer agent and metabolite combinations. Such analyses are expected to provide insight into the beneficial or harmful effect(s) of metabolites towards parent drug activity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This Letter describes the novel radiosensitizing agents based on nucleoside base modification. In addition to the known 5-phenylselenide derivative, 5-methylselenide modified thymidine, which has a van der Waals radius smaller than the phenyl group, was newly synthesized. The similar monomer activity of 5-methylselenide derivative under oxidation condition was confirmed by NMR experiments. The cytotoxicity tests and radiosensitizing experiments of both compounds were carried out using the H460 lung cancer cell line. Both the 5-phenylselenide and the 5-methylselenide derivatives showed a relatively low toxicity to the cells. However, in combination with γ-radiolysis, both exerted good radiosensitizing effects to the lung cancer cell lines in vitro. This result confirms that 5-methylselenide modified thymidine could be a useful candidate as a potential radiosensitizing agent in vivo., (Copyright © 2011. Published by Elsevier Ltd.)

Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.

Nonciliated bronchiolar (Clara cells) are progenitor cells during development. During differentiation, they are more susceptible to injury by environmental toxicants metabolized by the cytochrome P450 monooxygenase system, and injury results in altered bronchiolar repair and development. Squamous cells and abnormal cuboidal epithelium persist into early adulthood. The hypothesis tested in this study was that the failure of bronchiolar epithelium to repair normally in neonates following injury is due to an inhibition of proliferation. A model of differential repair in rabbit kits was used. Proliferation was followed for 1 week post injury in rabbit kits treated with a single dose of the P450-mediated cytotoxicant 4-ipomeanol (IPO) at 7 days old (repair abnormal) and compared to rabbits treated with a single dose of IPO at 21 days old (repair normal). Proliferation was measured by the nuclear incorporation of 5-chloro-2'-deoxyuridine (CldU) within epithelium at the target site (terminal bronchiole). The repair pattern between the two age groups was histologically defined. There was no difference in the CldU labeling index during the week of repair between the two age groups, even though the bronchiolar epithelium did not return to normal in the animals treated at 7 days old. In summary, proliferation (through S-phase) is not inhibited during repair in neonatal rabbits treated with IPO at 7 days old compared to animals treated at 21 days old, and we conclude that other factors may be responsible for the altered repair in the young neonates injured by a P450-mediated cytotoxicant.

5-Formyluracil (5-foU) is a major oxidation product of thymine formed in yields comparable to that of 8-oxoguanine in DNA by ionizing radiation. Whereas the mutagenic effects of 8-oxoguanine are well understood, the investigation of the biological implications of 5-foU has so far been limited. Here we demonstrate that 5-formyl-2'-deoxyuridine (5-fodUrd) supplied to the growth medium of Escherichia coli induces several base substitutions at different frequencies at position 461 in the lacZ gene in the following order: A.T-->G.C>G.C-->A.T>G.C-->T.A>>A.T-->T.A>A.T-->C.G. No induction of G.C-->C.G transversions was observed. It is inferred that 5-fodUrd will be incorporated into the DNA during cell growth, forming mispairs with guanine, cytosine and thymine during replication. It, thus, appears that cell growth in the presence of 5-fodUrd may represent a good model for elucidating the cellular effects of 5-foU residues in DNA.

Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluracil (5-foU) as major products. Whereas 5-hmU appears to have normal base pairing properties, the biological effects of 5-foU are rather poorly characterised. Here, we show that the colony forming ability of Chinese hamster fibroblast (CHF) cells is greatly reduced by addition of 5-foU, 5-formyluridine (5-foUrd) and 5-formyl-2'-deoxyuridine (5-fodUrd) to the growth medium. There are no toxic effects of 5-fodUrd on cells defective in thymidine kinase or thymidylate synthetase, suggesting that the toxicity may be caused by 5-fodUrd phosphorylation and subsequent inhibition of thymidylate synthetase. Whereas 5-fodUrd was the most effective 5-foU derivative causing cell growth inhibition, the corresponding ribonucleoside 5-foUrd was more effective in inhibiting [3H]uridine incorporation in non-dividing rat nerve cells in culture, suggesting that 5-foUrd exerts its toxicity through interference with RNA rather than DNA synthesis. Addition of 5-foU and 5-fodUrd was also found to promote mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of CHF cells; 5-fodUrd being three orders of magnitude more potent than 5-foU. In contrast, neither 5-hmU nor 5-(hydroxymethyl)-2'-deoxyuridine induced HPRT mutations. The mutation induction indicates that 5-foU will be incorporated into DNA and has base pairing properties different from that of thymine. These results suggest that 5-foU residues, originating from incorporation of oxidised bases, nucleosides or nucleotides or by oxidation of DNA, may contribute significantly to the damaging effects of oxygen radical species in mammalian cells.

beta-5-o-Carboranyl-2'-deoxyuridine (D-CDU) is a nontoxic pyrimidine nucleoside analogue designed for boron neutron capture therapy of brain tumors. In vitro studies indicated that D-CDU accumulates to levels 92- and 117-fold higher than the extracellular concentration in rat 9L and human U-251 glioma cells, respectively, and persists for several hours at levels 5-fold higher than the extracellular concentration. Furthermore, D-CDU was not toxic to rats injected i.p. with up to 150 mg/kg. On the basis of these studies, D-CDU was evaluated as a neutron capture therapy agent using rats bearing stereotactically implanted intracranial 9L tumors at single i.p. doses of 30 mg/kg and 150 mg/kg of D-CDU (20% 10B enriched), given 2 h before irradiation with thermal neutrons. Boron concentrations in tumors 2 h after dosing were 2.3 +/- 1.6 and 7.4 +/- 1.3 micrograms boron/g tissue (mean +/- SD), corresponding to tumor/brain ratios of 11.5 +/- 3.6 and 6.8 +/- 2.0 micrograms boron/g tissue for the low and high doses, respectively. All untreated animals died within 28 days, whereas half survived at days 32, 55, and 38 for groups receiving neutrons only, 30 mg/kg D-CDU, and 150 mg/kg D-CDU, respectively. Odds ratios of all treatment groups differed significantly from the untreated group (P < 0.002; logrank test). The median survival time for the 30 mg/kg-treated group but not for the 150 mg/kg-treated group was significantly longer than for rats treated with neutrons only (P = 0.036), which may correlate with the decreased tumor selectivity for D-CDU observed at the higher dose. Additional pharmacodynamic studies are warranted to determine optimal dosing strategies for D-CDU.

Unlabelled: We investigated the distribution, stability and radiotoxicity of 125I-oligodeoxyribonucleotides (125I-ODN) in human fibrosarcoma HT-1080 cells to study the radiotoxic effects of the Auger electron emitter 125I delivered to the cells by ODN., Methods: We delivered 125I-ODN into the cells via complexing with a liposomal delivery system. To assess the intracellular distribution and stability of 125I-ODN delivered by the liposomal delivery system, we used autoradiography, fluorescent and confocal microscopy and electrophoresis. To study the radiotoxicity of the unbound 125I-ODN, we used a clonogenic assay. The radiotoxicity of 125I-ODN delivered by the liposomal delivery system was compared with that of freely diffusible 125I-antipyrine, membrane-excluded 125I-bovine serum albumin and DNA incorporated 125I-deoxyuridine (125I-UdR)., Results: Oligodeoxyribonucleotides accumulated in the cell nucleus within a few hours of incubation. On the basis of the number of decays at 37% survival, 125I-ODN are 2 times more radiotoxic than 125I-antipyrine, which is freely diffusible into cells, and 8 times more radiotoxic than 125I-bovine serum albumin, which remains outside cells. However, the radiotoxicity of unbound 125I-ODN is almost 3 orders of magnitude lower than that of DNA-incorporated 125I-UdR. The 125I-ODN are not significantly degraded by intracellular nucleases during the time of uptake incubation., Conclusion: The dramatic difference in radiotoxicity between 125I-ODN and 125I-UdR confirms that, despite the nuclear localization, 125I-ODN are not bound to or incorporated within the genomic DNA. Our data demonstrate that the radiotoxicity of Auger electron emitters is determined by the radiation dose delivered to nuclear DNA, not necessarily to the nucleus. Therefore, relatively high intracellular concentrations of unbound 125I-ODN can be achieved without causing significant cell death.

Long-term therapy of AIDS patients with 3'-azido-3'-deoxythymidine (AZT) remains of concern because of resulting hematopoietic toxicity. While the mechanism(s) of this toxicity remains elusive, alternative strategies are being developed to reduce these toxic effects, including combination therapy with nonmyelotoxic antihuman immunodeficiency virus drugs and/or administration of protective or rescue agents, including cytokines and growth factors. By using a particularly relevant human CD34+ liquid culture system, the unique profiles of dideoxynucleoside (ddN) toxicities to both proliferation and differentiation were demonstrated, with decreased potencies in the order of 3'-fluoro-3'-deoxythymidine (FLT) = 3'-amino-3'-deoxythymidine (AMT) = 2',3'-dideoxycytidine > AZT for inhibition of proliferation and in the order of FLT = AMT > AZT >> 2',3'-dideoxycytidine for inhibition of hemoglobin synthesis. Hemin selectively protected erythroid-lineage human burst-forming unit-erythroid cells from AZT- and AMT-induced inhibition but had no effect on FLT toxicity under similar conditions. Myeloid-lineage human CFU-granulocyte-macrophages were also not protected by hemin against all three ddN analogs. The simultaneous exposure of cells to hemin and AZT resulted in a complete protection of both cell proliferation and hemoglobin synthesis. In contrast, in reversal studies only the inhibition of the percentage of hemoglobin-synthesizing cells returned to control levels, but the inhibition of proliferation of cells previously exposed to AZT was not reversed by hemin. These studies further define the unique and multifactorial mechanism(s) of ddN-induced toxic effects during hematopoietic development of pluripotent stem cells and suggest that the use of hemin could be beneficial in alleviating the toxicity of certain ddN analogs.

3'-Aminocyanoborane-2', 3'-dideoxythymidine (VIIa) and 3'-aminocyanoborane-2', 3'-dideoxyuridine (VIIIb) were successfully synthesized. The thymidine derivative (VIIIa) was shown to be a potent cytotoxic agent in murine and selected human suspended and solid tumor cell lines. Compound VIIIa inhibited L-1210 leukemia DNA and RNA synthesis with the protein synthesis requiring a higher concentration of drug for inhibition within 60 min. The purine pathway appeared to be the major target of Compound VIIIa with inhibition of IMP dehydrogenase and dihydrofolate reductase activities. The compound affected metabolic enzyme activities in the pyrimidine pathway as well as the nucleoside kinase activities. The DNA molecule did not appear to be target of the 3'-aminocyanoborane-2', 3'-dideoxythymidine (VIIIa), in that there was no change in ct-DNA viscosity, thermal denaturation or absorption of nucleosides of DNA nor was there any L-1210 DNA strand scission or inhibition of L-1210 DNA topoisomerase II activity when compound VIIIa was incubated at 100 microM.

5-Phenethyl-2'-deoxyuridine (PEUdR) augmented 5-fluoro-2'-deoxyuridine (FUdR) cytotoxicity up to 100-fold in several human gastric cancer cell lines. PEUdR also potentiated 5-fluorouracil (5-FU) cytotoxicity about 5-fold. In contrast, PEUdR reversed 5-fluorouridine (FUR) cytotoxicity in all cell lines studied. PEUdR was not cytotoxic up to 200 microM. PEUdR inhibited the incorporation of [3H]thymidine and [14C]uridine into acid-insoluble fractions, and also inhibited uptake of [3H]thymidine into KATO III cells. Thus, PEUdR inhibits pyrimidine nucleoside transport and salvage enzymes, which potentiates the cytotoxicity of FUdR and reverses the effect of FUR in human gastric cancer cells. These results may contribute to more effective cancer chemotherapy with FUdR and 5-FU.

The antiviral effect of 5-isopropyl-2'-deoxyuridine (IPDU) on herpes viruses (HSV-1 and HSV-2) is based on the sensitivity of virus DNA to nucleases due to IPDU incorporation into the virus DNA and the resulting change in DNA conformation. Virus selectivity is based on virus induced thymidine kinase activity and on HSV induction of polymerase enzymes catalyzing the incorporation. Hevizos ointment containing IPDU has the following advantageous pharmacokinetic properties: high accumulation in the skin, high resistance to phosphorylases and other metabolizing enzymes.

The specific objective was to find what processes are responsible for the mutagenicity of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU), which is a product of ionizing radiation, and what role transition metal ions play in those processes. We found that HPMdU is a more potent mutagen than its decomposition products 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU) in the Salmonella typhimurium strains tested, with the TA100 strain being the most sensitive. HMdU exerted intermediate mutagenicity and FdU was the weakest of the three compounds. At 50 nmoles/plate, HPMdU increased the number of revertants by 4-fold, whereas 1000 nmoles HMdU was required to enhance the number of revertants by 5-fold. Pretreatment of TA100 with o-phenanthroline, a membrane-permeable Fe and Cu chelator, caused an increase in mutagenicity of the low HPMdU doses but inhibited that of the 50 nmoles HPMdU/plate, while desferal, a membrane-impermeable Fe chelator, had virtually no effect. Azide (a catalase inhibitor) enhanced HPMdU mutagenicity, whereas 3-amino-1,2,4-triazole (a catalase and peroxidase inhibitor) and ammonium formate (a hydroxyl radical scavenger) were protective. Preincubation of TA100 cells with 20 and 40 nM HPMdU caused dose-dependent formation of the oxidized DNA base derivatives HMdU, thymidine glycol and 8-hydroxyl-2'-deoxyguanosine (8-OHdG), known hydroxyl radical-mediated oxidation products. Cumulatively, these results suggest that the genetic effects of HPMdU are due to its hydroperoxide moiety, which upon reacting with Fe generates hydroxyl radicals that in turn oxidize neighboring bases in cellular DNA. This also may be a mechanism by which ionizing radiation exerts its long-term effects.

Syntheses of the two diastereomers (5a and 6a) of 5-(2,2-dichlorocyclopropyl)-, and of the four diastereomers (7a-10a) of 5-(2-chlorocyclopropyl)-2'-deoxyuridine are described. These, and corresponding diastereomers (5b and 6b; 7b-10b) of 5-(2,2-dibromocyclopropyl)- and 5-(2-bromocyclopropyl)-2'-deoxyuridine (prepared in an earlier investigation) were examined for antiviral and cytotoxic activity, in comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine (FDU). 5-[(1R,2R)-2-Chlorocyclopropyl]-2'-deoxyuridine (9a) was the most active antiviral agent (IC50 = 25 micrograms/ml) against herpes simplex virus type 1 (HSV-1) relative to BVDU (IC50 = 0.082 microgram/ml). Compounds having the R configuration at the C-1 and/or C-2 positions of the 5-[2,2-dichloro(or 2-chloro)cyclopropyl] substituent exhibited the most potent antiviral activity. The cytotoxic activities of the 5-(2,2-dihalocyclopropyl)- (5-6a and b) and 5-(2-halocyclopropyl)- diastereomers (7-10a and b) were dependent upon both the configuration of the C-1 and/or C-2 cyclopropyl carbons and the nature of the halogeno (Cl, Br) substituent. 5-[(1R)-2,2-Dichlorocyclopropyl]-2'-deoxyuridine (6a) was the most active cytotoxic compound in the CCRF-CEM (IC50 = 17 microM) and HL-60 (IC50 = 64 microM) screens relative to FDU, which exhibited IC50 values of 4.7 x 10(-3) and 77 microM in these respective screens.

An aqueous solution of calf thymus DNA was irradiated by 60Co gamma-rays and modified nucleosides produced in DNA were analyzed by high-pressure liquid chromatography coupled with a photodiode array UV detector. A new product with UV absorption maxima at 230 nm and 280 nm was observed. The structure of this compound was proposed to be 5-formyldeoxyuridine (f5dU) based on the mass spectrum of its trimethylsilyl derivative (M+, m/z472) and the structure was confirmed by chemical synthesis. The yield of f5dU (2.4/10(4) dT/krad) in DNA was of roughly the same order as that of 8-hydroxydeoxyguanosine and 5-hydroxymethyldeoxyuridine. Free f5dU was mutagenic to Salmonella typhimurium strain TA102: therefore f5dU incorporated into DNA may induce mutations.

Various 5-substituted pyrimidine 2'-deoxyribosides with anti-herpes activity were investigated for their genotoxic activity. 5-Iodo-2'-deoxycytidine (IDC), 5-(2-chloroethyl)-2'-deoxycytidine (CEDC), 5-(3-chloropropyl)-2'-deoxyuridine (CPDU), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 5-ethyl-2'-deoxyuridine (EDU), 2'-deoxyuridine (DU) and 2'-deoxythymidine (DT) were non-mutagenic in Salmonella typh. as well as in V79 Chinese hamster cells, 5-Iodo-2'-deoxyuridine (IDU) was moderately mutagenic and 5-(2-chloroethyl)-2'deoxyuridine (CEDU) was highly mutagenic in V79 cells; neither IDU nor CEDU were mutagenic in the bacterial assay. None of the compounds induced unscheduled DNA synthesis in primary rat hepatocytes. In addition, antimutagenic effects of 2'-deoxyuridines were discovered: in V79 cells, BVDU, EDU, DU and DT prevented the mutagenicity induced by CEDU; in these cells EDU also inhibited the mutagenicity induced by MNNG. In primary rat hepatocytes, IDU and EDU inhibited the induction of unscheduled DNA synthesis induced by MNNG, DMBA or UV-light. The compounds were inactive at inducing differentiation in hematopoietic cells. The significance of these data, particularly with regard to the use of 5-substituted 2'-deoxyuridines in anti-herpes therapy, is discussed.

We investigated the toxicity of intravitreal zinc sulfate, zinc gluconate, and ethyldeoxyuridine (EDU) in albino rabbits. Various concentrations of EDU were added to the infusion solution during pars plana vitrectomy. Retinal changes were observed by light microscopy after intravitreal injections containing 20 micrograms of zinc gluconate, all concentrations of zinc sulfate, and 400 micrograms of EDU. No histologic or electroretinographic alterations occurred with doses of 15 micrograms or less of zinc gluconate or 200 micrograms or less of EDU. When added to the vitrectomy solution, concentrations of 20 micrograms/mL or less of EDU appeared nontoxic to the rabbit retina.

Several 5-substituted 2'-deoxyuridine (dUrd) analogues, known for their anti-viral or anti-tumour potentials, were evaluated for their ability to induce sister-chromatid exchange (SCE) in human fibroblasts and lymphocytes. While the selective anti-herpes agents (E)-5-(2-bromovinyl)-dUrd, (E)-5-(2-chlorovinyl)-dUrd, (E)-5-(2-iodovinyl)-dUrd, (E)-5-(3,3,3-trifluoro-1-propenyl)-dUrd and (E)-5-(1-propenyl)-dUrd did not induce SCE unless their concentration was about 1000-5000-fold greater than that required to inhibit herpes-virus replication, several other dUrd analogues such as 5-vinyl-dUrd, 5-ethynyl-dUrd, 5-formyl-dUrd, 5-hydroxymethyl-dUrd and 5-trifluoromethyl-dUrd induced SCE at a concentration closely corresponding to those required to inhibit either virus replication or tumour-cell growth. Only 2 compounds, 5-fluoro-dUrd and 5-nitro-dUrd (5'-monophosphate), inhibited tumour-cell growth at a concentration well below that required for increased SCE induction. The ability of a given dUrd analogue to induce SCE may be related to its incorporation into DNA or, alternatively, may depend on an interaction with 5-bromo-dUrd, which is included in the tests to visualize SCE and which in itself produces SCE.

5-Ethyl-2'-deoxyuridine (5-ethyl-dUrd), an analog of thymidine, was evaluated for its capacity to inhibit herpes simplex virus (HSV) replication in vitro and in vivo. The 50% effective dose concentration of 5-ethyl-dUrd for HSV types 1 and 2 (HSV-1 and -2) cultured in Vero cells was 6 and 9 mug/ml, respectively. Levels of 5-ethyl-dUrd 14-fold in excess of the 50% effective dose for HSV-1 did not inhibit the formation of confluent monolayers by Vero cells, suggesting that the compound was not cytotoxic or inhibitory for mammalian cells. In vivo studies showed that 5-ethyl-dUrd was effective in significantly reducing mortality when administered to young adult mice after subcutaneous infection with HSV-2. Intraperitoneal and intravenous inoculation of drug (250 mg/kg per day) resulted in a 50% survivor rate at 15 days. Comparative studies with adenine arabinoside at 250 mg/kg per day gave a 40% survivor rate. Intramuscular injection of 5-ethyl-dUrd at a concentration as high as 2,000 mg/kg per day for 10 days was well tolerated by uninfected animals, and HSV-2-infected mice treated at this dosage had a 100% survival rate. Treatment with 5-ethyl-dUrd at a concentration of 500 mg/kg per day significantly increased the mean survival times of HSV-1- and HSV-2-infected mice immunosuppressed by irradiation; however, the fatal course of the infection was not altered. Assay for virus in tissues showed that 5-ethyl-dUrd treatment delayed progression of the infection into the central nervous system, indicating suppression of virus replication in the tissues.

The adenosine deaminase-resistant purine deoxynucleoside 2-chloro-2'-deoxyadenosine (CdA) is markedly toxic in vitro to nondividing and proliferating normal human lymphocytes and to many leukemia cell specimens. The CdA is also effective against mouse L1210 leukemia in vivo. The present investigations have examined the pharmacology, chemotherapeutic activity, and toxicity of CdA in nine patients with advanced hematologic malignancies refractory to conventional therapy. When administered by continuous intravenous infusion, the deoxyadenosine analog was well tolerated. As monitored by radioimmunoassay, plasma CdA levels rose gradually during the infusions. The CdA was not deaminated significantly. In all patients with leukemia, the CdA lowered the blast count by at least 50%. In one patient with a T-cell leukemia-lymphoma, and in another patient with chronic myelogenous leukemia in blast crisis, the CdA infusion eliminated all detectable blasts from the blood and bone marrow. In a patient with a diffuse lymphoma complicated by severe autoimmune hemolytic anemia, CdA treatment quickly terminated the hemolytic process. Bone marrow suppression represented the dose-limiting toxicity, and was related to plasma CdA levels, cumulative drug dosage, and the rapid release of CdA that accompanied tumor cell lysis.

Previous results from this laboratory have shown that thymidylate deprivation results in dramatic elevation of intracellular dUTP and incorporation of dUMP into DNA. The goal of the present studies was to determine whether the latter changes may play a part in the associated cytotoxicity ("thymineless death"), which is ordinarily assumed to be a direct result of reduced intracellular dTTP. The approach used here was to increase intracellular dUTP without allowing dTTP to diminish and observe the effects on cell viability. dUMP pools were expanded by exposure of cells to deoxyuridine [in cell growth medium containing hypoxanthine, methotrexate, and thymidine (HAT medium)], resulting in accumulation of dUTP to levels that approached those of dTTP, which were at, or higher than, the levels in untreated cells. In conjunction with this the cells became nonviable, and newly synthesized DNA was fragmented, both of which occur with thymidylate depletion and, we assume, result from the active process of excision repair at the many uracil-containing sites in DNA. The results indicate that, although the relative importance of low dTTP remains unknown, elevated dUTP can account for the cytotoxicity caused by thymidine starvation. Most of the "dTTP" measured by the DNA polymerase assay in cells treated with methotrexate (MTX) (plus purine supplement) was, in fact, dUTP, which may explain some previous observations of only modest depression of dTTP in cells treated with MTX or similarly acting drugs.

The production of lung adenomas in strain A mice following multiple injections of 17 alkyl halides and of 3 base analogs was investigated. A slight but significant increase in the average number of lung tumors per mouse was noted following the administration of methyl iodide, n- and i-propyl iodide, sec- and tert-butyl chloride, i-, sec-, and tert-butyl bromide, and n- and sec-butyl iodide. The administration of comparable doses of ethyl bromide, ethyl iodide, n-butyl chloride, benzyl chloride, and 1-chloromethylnaphthalene to mice resulted in no significant increase in the frequency of lung tumors over that seen in vehicle-treated control mice. n-Butyl bromide and tert-butyl iodide similarly appeared to have no significant effect on the lung tumor frequency, but these compounds were too toxic to be tested at the high dosages used with the other alkyl halides. 5-Iodo-, 5-bromo-, and 5-fluorodeoxyuridine also appeared to have no significant effect on the lung tumor frequency. These results indicate that a high proportion of low-molecular-weight alkyl halides may be weakly carcinogenic and provide evidence supporting an electrophilic hypothesis of carcinogenesis.

EM9 is a mutagen-sensitive CHO cell whose phenotype resembles that of normal CHO cells exposed to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis. This phenotype suggested that EM9 might be defective in poly(ADP-ribose) metabolism, but we now cannot find any abnormality in the synthesis or in the degradation of poly(ADP-ribose) in permeabilized EM9 cells. Thus the effects of 3-aminobenzamide on wild-type cells may be due to the inhibition of processes other than poly(ADP-ribose) synthesis. 3-Aminobenzamide enhances the cytotoxicity of EMS toward EM9 and control cells to the same degree.

The (chloroethyl)nitrosourea analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine, 3'-[3-(2-chloroethyl)-3-nitrosoureido]-2',3'-dideoxyuridine (3'-CdUNU, 7) and 3'-[3-(2-chloroethyl)-3-nitrosoureido]-2,3'-dideoxy-5-fluorouridine (3'-CFdUNU, 8), have been synthesized by treatment of the corresponding 3'-amino nucleosides with chloroethyl isocyanate, followed by nitrosation of the resulting ureas. Nucleoside nitrosoureas 7 and 8 exhibited marked anticancer activity against L1210 leukemia in tumor-bearing mice. At an optimum dosage level of 40 mg/kg, 7 and 8 produced 90% and 60% "cures" (greater than 60-day survivors), respectively. The structure-activity relationships are discussed.

The nuclear enzyme, poly(ADP-ribose) synthetase is involved in the repair of damaged DNA. We report here the results obtained with 3-aminobenzamide (3AB), an inhibitor of this enzyme, on induced biological effects. 3AB increases the frequency of chromosomal aberrations induced by DMS, EMS, ENU, bleomycin and CldUrd. The magnitude of the effect is dependent on the type of chemical used, the combinations with DMS and EMS being the most potent ones. No potentiation was observed after treatment of cells with MMC. Mutation frequencies were determined on the HPRT locus and showed that 3AB did not increase the frequency of gene mutations induced by EMS, ENU and CldUrd. Cell-cycle progression is affected when cells are grown in medium containing CldUrd and 3AB, primarily when the inhibitor is present during the second cell cycle when substituted DNA becomes replicated. The extent of the effect depends on the amount of analogue incorporated and is independent of the presence of the analogue in the medium during the second cell cycle. Analysis of chromosomal aberrations in delayed G2 cells with the aid of the premature chromosome-condensation technique revealed numerous aberrations after incorporation of CldUrd and treatment with 3AB.

5-Trifluoromethyl-2'-deoxyuridine (F(3)dThd) was evaluated for its neurotoxicity and for its ability to increase the life span of mice injected intracerebrally with herpes simplex virus type 1 (HSV-1) and F(3)dThd simultaneously. F(3)dThd showed no neurotoxicity at the highest concentration tested (100 mg/kg). Mice injected intracerebrally with HSV-1 died within 5 days postinfection. However, all mice injected concurrently with HSV-1 and 100 mg of F(3)dThd per kg lived through the termination of the experiment (60 days). Protection of mice from HSV-1 encephalitis by F(3)dThd has been shown to be dose dependent, with 100, 75, 50, and 25 mg of F(3)dThd per kg yielding a survival rate of 100, 90, 50, and 10%, respectively. HSV-1 titers in mouse brains receiving HSV-1 and 100 mg of F(3)dThd per kg concurrently were 100- to 1,000-fold lower at 2 to 4 days postinfection than control mice receiving HSV-1 alone. F(3)dThd was shown not to stimulate interferon production. Encephalitis caused by a ribonucleic acid virus, encephalomyocarditis virus, was not modified by F(3)dThd treatment.

5-Ethyl-, 5-vinyl-, 5-propyl-, and 5-allyl-2'-deoxyuridine (dUrd) had antiviral activity against herpes simplex type 1 and type 2 grown in HeLa TK(-) cells, in the order 5-vinyl-dUrd, 5-ethyl-dUrd, 5-propyl-dUrd, 5-allyl-dUrd, but they were inactive against a TK(-) mutant of herpes simplex type 1. The antiviral activity of these compounds could be partially reversed by thymidine. Except for 5-vinyl-dUrd, they were not toxic to WI-38 and HeLa TK(-) cells at a concentration of 25 muM. All four analogues inhibited the growth of herpes simplex type 1-transformed HeLa TK(-) cells at a concentration of 1 muM.

Treatment of 5-mercaptomethyluracil (I) with trimethylsilyl chloride in the presence of triethylamine gave 2,4,5-tris-(trimethylsilyl)-5-mercaptomethyluracil (II) which, upon coupling with 2-deoxy-3,5-di-O-(p-toluoyl)-D-erythro-pentofuranosyl chloride, furnished as anomeric mixture of fully substituted 2'-deoxy ribonucleosides. The nucleoside with beta configuration (III) was predominantly formed and was isolated as a crystalline solid. The free nucleoside IV was obtained by removal of blocking groups by sodium methoxide catalyzed deacylation, deionization under reducing atmosphere, and chromatography on neutral alumina. IV is oxidized to the corresponding disulfide V in solution in the absence of thiols. IV was found to be markedly inhibitory against the herpes virus of infectious bovine rhinotracheitis (IBR). Against this virus, IV was found to be as potent as 5-iododeoxyuridine and cytosine arabinoside when added 18 hr before virus infection.

Using an in vitro system we measured the corneal epithelial cytotoxicity and the antiviral activity of the antiviral agents idoxuridine (IDU), trifluridine (TFT), ethyldeoxyuridine (EDU), and (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU). Confluent rabbit corneal epithelial cell cultures were established, and the antiviral agents were added for 5, 30, or 60 min at a range of concentrations including that used clinically (IDU 0.1%, TFT 1.0%, BVDU 0.1%, EDU 2.0%). Twelve hour [3H]thymidine incorporation then was measured and expressed as % inhibition of control cultures. In separate experiments confluent corneal epithelial cell monolayers were inoculated with 10(4) plaque forming units (PFU) of HSV type 1 (McKrae strain) for 1 h, and IDU 0.1%, TFT 1.0%, and BVDU 0.1% were added to the culture for determination of PFU inhibition. Significant dose-, but not time-dependent, toxicity was observed at the clinical concentrations of IDU, TFT, and EDU. Toxicity was absent for BVDU. TFT and IDU were the most toxic, and EDU was of intermediate toxicity. IDU, TFT, and BVDU showed significant antiviral activity in this corneal epithelial cell culture system (TFT greater than BVDU greater than IDU). The results of this in vitro study paralleled the findings of previous in vivo corneal epithelial toxicity studies of IDU, TFT, and BVDU. Our data, however, suggest that EDU has a potential for clinical toxicity and further studies are recommended. Our model may be useful in the future toxicologic study of new antiviral agents.

The alkaline elution of bromodeoxyuridine-containing (BrdUrd) DNA and chlorodeoxyuridine-containing ( CldUrd ) DNA was studied in two CHO lines, the parental AA8 and a mutant line, EM9 , which has a defect in repairing strand breaks and a 12-fold elevated baseline frequency of SCE. BrdUrd-DNA was found to have alkali-labile sites as well as direct breaks, neither of which were increased significantly by prior treatment of AA8 cells with an inhibitor (benzamide) or poly(adenosine diphosphoribose) polymerase. CldUrd -DNA, which gives higher frequencies of SCEs than BrdUrd-DNA, had more strand breaks than BrdUrd-DNA in AA8 cells after treatment with benzamide, while without benzamide there was no difference. The accumulation of breaks in CldUrd -DNA by benzamide was shown to occur rapidly, to reach a maximum by 90 min, and to be readily reversible after benzamide removal. Under all conditions, EM9 cells had more strand breaks than AA8 . These observed differences in strand breaks were not due to differences in incorporation efficiencies. For the different halogenated pyrimidines and cell types, there was a good correlation between the number of strand breaks and reduction in plating efficiencies.

Toxic effects of 5-methoxymethyl-2'-deoxyuridine (MMdUrd), a drug with antiviral activity against herpes simplex virus, were investigated in Chinese golden hamsters. Pathological, hematological, and clinical chemistry parameters were studied. Systemic toxicity was not observed in hamsters after intraperitoneal (ip) administration of MMdUrd in single doses up to 3000 mg/kg and in repeated dosages of 600 mg/kg daily for 15 days. No gross or microscopic lesions related to MMdUrd treatment were observed in the tissues examined from the animals of subchronic toxicity study. Administration of a single dose of 6000 mg/kg of MMdUrd ip caused cloudy swelling, increased mitotic figures, varied nuclear sizes, and scattered coagulation necrosis of hepatocytes on the second day after drug administration. These hepatic lesions were not observed in animals sacrificed on the fourth and sixth days postadministration of MMdUrd. Microscopic examination of marrow smears from control and MMdUrd-treated animals revealed no differences in morphological features of hematopoiesis, nor were significant differences observed between treated and control animals for hematological parameters. Increased concentrations of alanine amino transferase were observed on the first and second day in animals, given a single dose of 6000 mg/kg of MMdUrd. Increases in sister chromatid exchanges and the number of azaguanine-resistant mutants were observed after exposure to a high concentration of MMdUrd (1024 micrograms/ml).

The metabolic consequences of deoxyuridine treatment in four cultured human lymphoblast lines (CCRF-CEM, RPMI-8402, JM, and BALM) were studied by cell growth experiments, flow cytometry, and measurement of 2'-deoxyribonucleoside triphosphate (dNTP) levels. DNA perturbations occurred in all lymphoblast lines, but there was no significant impairment of RNA synthesis. The DNA perturbations in CCRF-CEM, RPMI-8402, and BALM cells reflected inhibition of DNA synthesis, and the associated dNTP changes were consistent with ribonucleotide reductase inhibition or, specifically in BALM cells, with DNA alpha-polymerase inhibition. JM cells treated with an intermediate concentration of deoxyuridine developed a block at the G1/S boundary which was deoxyuridine concentration-dependent, but not specific for deoxyuridine (it was also seen with thymidine treatment) and not related to DNA synthesis inhibition. There were no idiosyncratic dNTP effects accompanying the G1/S boundary block, and the responsible metabolic mechanism remains to be determined.