Single-Molecule DNA Fiber Analyses to Characterize Replication Fork Dynamics in Living Cells.

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.

Phase I and Pharmacology Study of Ropidoxuridine (IPdR) as Prodrug for Iododeoxyuridine-Mediated Tumor Radiosensitization in Advanced GI Cancer Undergoing Radiation.

PURPOSE: Iododeoxyuridine (IUdR) is a potent radiosensitizer; however, its clinical utility is limited by dose-limiting systemic toxicities and the need for prolonged continuous infusion. 5-Iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is an oral prodrug of IUdR that, compared with IUdR, is easier to administer and less toxic, with a more favorable therapeutic index in preclinical studies. Here, we report the clinical and pharmacologic results of a first-in-human phase I dose escalation study of IPdR + concurrent radiation therapy (RT) in patients with advanced metastatic gastrointestinal (GI) cancers. PATIENTS AND METHODS: Adult patients with metastatic GI cancers referred for palliative RT to the chest, abdomen, or pelvis were eligible for study. Patients received IPdR orally once every day × 28 days beginning 7 days before the initiation of RT (37.5 Gy in 2.5 Gy × 15 fractions). A 2-part dose escalation scheme was used, pharmacokinetic studies were performed at multiple time points, and all patients were assessed for toxicity and response to Day 56. RESULTS: Nineteen patients were entered on study. Dose-limiting toxicity was encountered at 1,800 mg every day, and the recommended phase II dose is 1,200 mg every day. Pharmacokinetic analyses demonstrated achievable and sustainable levels of plasma IUdR ≥1 µmol/L (levels previously shown to mediate radiosensitization). Two complete, 3 partial, and 9 stable responses were achieved in target lesions. CONCLUSIONS: Administration of IPdR orally every day × 28 days with RT is feasible and tolerable at doses that produce plasma IUdR levels ≥1 µmol/L. These results support the investigation of IPdR + RT in phase II studies.

Toxicology and pharmacokinetic study of orally administered 5-iodo-2-pyrimidinone-2'deoxyribose (IPdR) x 28 days in Fischer-344 rats: impact on the initial clinical phase I trial design of IPdR-mediated radiosensitization.

PURPOSE: A toxicology and pharmacokinetic study of orally administered (po) IPdR (5-3iodo-2-pyrimidinone-2'deoxyribose, NSC-726188) was performed in Fischer-344 rats using a once daily (qd) x 28 days dosing schedule as proposed for an initial phase I clinical trial of IPdR as a radiosensitizer. METHODS: For the toxicology assessment, 80 male and female rats (10/sex/dosage group) were randomly assigned to groups receiving either 0, 0.2, 1.0 or 2.0 g kg(-1)day(-1) of po IPdR x 28 days and one-half were observed to day 57 (recovery group). Animals were monitored for clinical signs during and following treatment with full necropsy of one-half of each dosage group at day 29 and 57. For the plasma pharmacokinetic assessment, 40 rats (10/sex/dosage group) were randomly assigned to groups receiving either 0.2 or 1.0 g kg(-1)day(-1) of po IPdR x 28 days with multiple blood samplings on days 1 and 28 and single blood sampling on days 8 and 15. RESULTS: No drug-related deaths occurred. Higher IPdR doses resulted in transient weight loss and transient decreased hemoglobins but had no effect on white cells or platelets. Complete serum chemistry evaluation showed transient mild decreases in total protein, alkaline phosphatase, and serum globulin. Necropsy evaluation at day 29 showed minimal to mild histopathologic changes in bone marrow, lymph nodes and liver; all reversed by day 59. There were no sex-dependent differences in plasma pharmacokinetics of IPdR noted and the absorption and elimination kinetics of IPdR were found to be linear over the dose range studied. CONCLUSIONS: A once-daily dosing schedule of po IPdR for 28 days with doses up to 2.0 g kg(-1)day(-1) appeared to be well tolerated in Fischer-344 rats. Drug-related weight loss and microscopic changes in bone marrow, lymph nodes and liver were observed. These changes were all reversed by day 57. IPdR disposition was linear over the dose range used. However, based on day 28 kinetics it appears that IPdR elimination is enhanced following repeated administration. These toxicology and pharmacokinetic data were used when considering the design of our initial phase I trial of po IPdR as a clinical radiosensitizer.

Bioassay of alkyl halides and nucleotide base analogs by pulmonary tumor response in strain A mice.

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.

An in vivo comparison of oral 5-iodo-2'-deoxyuridine and 5-iodo-2-pyrimidinone-2'-deoxyribose toxicity, pharmacokinetics, and DNA incorporation in athymic mouse tissues and the human colon cancer xenograft, HCT-116.

5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) was recently reported to be converted to 5-iodo-2'-deoxyuridine (IUdR) by an aldehyde oxidase, most concentrated in liver tissue. We questioned whether IPdR could be used as a p.o. hepatotropic prodrug to increase the percentage of IUdR-DNA incorporation into liver tumors compared to normal liver with acceptable systemic toxicity. Athymic nude mice with human colon cancer (HCT-116) xenograft tumors as liver metastases and s.c. flank tumors received daily p.o. boluses (via gastric tubes) of IUdR or IPdR for 6 days. The maximum tolerated dose of IUdR was 250 mg/kg/day and was associated with a > 10% weight loss and a high percentage of IUdR-DNA incorporation (> 5%) into normal bone marrow and intestine. In contrast, animals tolerated escalating doses of IPdR to 1 gm/kg/day without weight loss and with less (1.5-4%) IUdR-DNA incorporation in normal tissues. Pharmacokinetic analysis of p.o. IPdR showed peak plasma levels of IPdR and IUdR within 15-45 min, suggesting efficient conversion of IPdR to IUdR. Aldehyde oxidase activity was found in normal liver tissue but not in other normal or tumor tissues. Additionally, we found a 2-3 times greater percentage of IUdR-DNA incorporation in tumor with IPdR than IUdR at the highest doses used. However, no differential effect in the percentage of IUdR-DNA incorporation was noted between liver metastases and s.c. tumors with either IPdR or IUdR. We conclude that p.o. IPdR offers a greater therapeutic index for tumor incorporation (and presumably radiosensitization) than a similar schedule of IUdR.

Fluorodeoxyuridine improves imaging of human glioblastoma xenografts with radiolabeled iododeoxyuridine.

Use of radiolabeled nucleotides for tumor imaging is hampered by rapid in vivo degradation and low DNA-incorporation rates. We evaluated whether blocking of thymidine (dThd) synthesis by 5-fluoro-2'-deoxyuridine (FdUrd) could improve scintigraphy with radio-dThd analogues, such as 5-iodo-2'-deoxyuridine (IdUrd). We first show in vitro that coincubation with FdUrd substantially increased incorporation of [125I]IdUrd and [3H]dThd in the three tested human glioblastoma lines. Flow cytometry analysis showed that a short coincubation with FdUrd (1 h) produces a signal increase per labeled cell. We then measured biodistribution 24 h after i.v. injection of [125I]IdUrd in nude mice s.c. xenografted with the three glioblastoma lines. Compared with animals given [125I]IdUrd alone, i.v. preadministration for 1 h of 10 mg/kg FdUrd increased the uptake of [125I]IdUrd in the three tumors 4.8-6.8-fold. Compatible with previous reports, there were no side effects in mice observed for 2 months after receiving such a treatment. The tumor uptake of [125I]IdUrd was increased < or =13.6-fold when FdUrd preadministration was stepwise reduced to 1.1 mg/kg. Uptake increases remained lower (between 1.7- and 5.8-fold) in normal proliferating tissues (i.e., bone marrow, spleen, and intestine) and negligible in quiescent tissues. DNA extraction showed that 72-80% of radioactivity in tumor and intestine was bound to DNA. Scintigraphy of xenografted mice was performed at different times after i.v. injection of 3.7 MBq [125I]IdUrd. Tumor detection was significantly improved after FdUrd preadministration while still equivocal after 24 h in mice given [125I]IdUrd alone. Furthermore, background activity could be greatly reduced by p.o. administration of KClO4 in addition to potassium iodide. We conclude that FdUrd preadministration may improve positron or single photon emission tomography with cell division tracers, such as radio-IdUrd and possibly other dThd analogues.

Fluorodeoxyuridine-mediated modulation of iododeoxyuridine incorporation and radiosensitization in human colon cancer cells in vitro and in vivo.

A study was conducted to assess the potential of 5-fluoro-2'-deoxyuridine (FdUrd) to increase the incorporation and radiosensitizing properties of 5-iodo-2'-deoxyuridine (IdUrd) using HT29 human colon cancer cells both in vitro and in nude mice bearing these tumors as xenografts. The purpose of this study was to assess (a) whether FdUrd could increase IdUrd efficacy using clinically achievable concentrations of drugs; (b) the relationships among radiosensitization, DNA damage and repair, and analogue incorporation; and (c) whether FdUrd improved the selectivity of IdUrd incorporation into tumor cells compared to normal tissues. It was found that FdUrd, at clinically achievable concentrations (1-100 nM), significantly increased IdUrd incorporation under all conditions but particularly when the IdUrd concentration was less than or equal to 10 microM. FdUrd increased IdUrd-mediated radiosensitization in proportion to the increase in IdUrd incorporation. FdUrd potentiated the ability of IdUrd to increase radiation-induced DNA double-strand breaks and to slow their repair. When IdUrd alone (100 and 200 mg/kg/day) was infused into nude mice bearing tumors, the extent of thymidine replaced in the tumor was 1.6 +/- 0.4 (mean +/- SE) and 2.5 +/- 0.4%, respectively. The combination of FdUrd (0.1 mg/kg/day) and IdUrd (100 mg/kg/day) increased the incorporation in the tumor to 5.3 +/- 0.9% with less toxicity than resulted from the use of 200 mg/kg/day of IdUrd alone. These data show that FdUrd is an effective biomodulator, because, for the same extent of normal tissue incorporation, the combination of IdUrd and FdUrd produces significantly greater incorporation into the tumor compared to the use of IdUrd alone. Furthermore, they suggest that the regional application of FdUrd with IdUrd, either through the use of regional infusions or in combination with focused irradiation, could potentially improve the outcome of treatment of localized gastrointestinal cancer.

Inhibition of base excision repair potentiates iododeoxyuridine-induced cytotoxicity and radiosensitization.

5-Iodo-2'-deoxyuridine (IdUrd) is a halogenated thymidine analogue recognized as an effective in vitro and in vivo radiosensitizer in human cancers. IdUrd-related cytotoxicity and/or radiosensitization are correlated with the extent of IdUrd-DNA incorporation replacing thymidine. IdUrd cytotoxicity and radiosensitization result, in part, from induction of DNA single-strand breaks (SSB) with subsequent enhanced DNA double-strand breaks leading to cell death. Because base excision repair (BER) is a major DNA repair pathway for SSB induced by chemical agents and ionizing radiation, we initially assessed the role of BER in modulating IdUrd cytotoxicity and radiosensitization using genetically matched Chinese hamster ovary cells, with (AA8 cells) and without (EM9 cells) XRCC1 expression. XRCC1 plays a central role in processing and repairing SSBs and double-strand breaks. We found that EM9 cells were significantly more sensitive than parental AA8 cells to IdUrd alone and to IdUrd + ionizing radiation. The EM9 cells also demonstrate increased DNA damage after IdUrd treatment as evaluated by pulse field gel electrophoresis and single cell gel electrophoresis (Comet Assay). BER-competent EM9 cells, which were stably transfected with a cosmid vector carrying the human XRCC1 gene, showed responses to IdUrd similar to AA8 cells. We also assessed the role of methoxyamine, a small molecule inhibitor of BER, in the response of human colon cancer cells (HCT116) to IdUrd cytotoxicity and radiosensitization. Methoxyamine not only was able to increase IdUrd cytotoxicity but also increased the incorporation of IdUrd into DNA of HCT116 human colon cancer cells leading to greater radiosensitization. Thus, a genetic or biochemical impairment of BER results in increased IdUrd-induced cytotoxicity and radiosensitization in mammalian cells.

In vivo modulation of iododeoxyuridine metabolism and incorporation into cellular DNA by 5'-amino-5'-deoxythymidine in normal mouse tissues and two human colon cancer xenografts.

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.

Clinical pharmacology of 5-iodo-2'-deoxyuridine and 5-iodouracil and endogenous pyrimidine modulation.

We describe the clinical pharmacology and metabolism of 5-iodo-2'-deoxyuridine (IdUrd) during and after a 12-hour infusion. The kinetics of IdUrd were linear between 250 and 1200 mg/m2. The plasma IdUrd concentration reached steady state in less than 1 hour. Total body clearance of IdUrd was 750 ml/min/m2 and the disappearance t1/2 at the end of the infusion was less than 5 minutes. The primary metabolite, 5-iodouracil (IUra), did not reach steady state during the infusion. At the end of the 1200 mg/m2 infusion, the maximum plasma IUra concentration was 100 mumol/L, or about 10 times the simultaneous IdUrd plasma concentration. During the infusion there was at least a fifty- to 100-fold increase in uracil and thymine plasma concentrations. After the infusion, IUra disappearance from plasma was nonlinear, with an apparent Michaelis constant of 30 mumol/L. Plasma uracil and thymine levels slowly decreased after the IdUrd infusion until IUra fell to less than 30 mumol/L. There was subsequently a parallel and more rapid decrease in the plasma concentrations of uracil and thymine. Uridine, 2'-deoxyuridine, and thymidine plasma levels did not change significantly as a result of IdUrd therapy. These changes in endogenous pyrimidine pools are consistent with competitive inhibition of dihydrouracil dehydrogenase by IUra. An in vitro human bone marrow assay was used to determine the relative toxicity of IdUrd and IUra. Although exposure to IUra was tenfold higher than that to IdUrd, IdUrd was at least 100 times more cytotoxic to marrow cells.

Studies on the microcytotoxicity test. III. Comparison of [75Se]selenomethionine with [3H]proline, Na2, 51CrO4 and [125I]iododeoxyuridine for pre-labelling target cells in long-term cytotoxicity tests.

Four intracellular radioisotope labels, [3H]proline, Na2 51CrO4, [75Se]selenomethionine and [125I]iododeoxyuridine, were evaluated for use in a pre-labelling long-term microcytotoxicity assay for cell-mediated immunity. Adherent rat tumour cells established in tissue culture were used as targets and the basic variables studied were labelling efficiency, toxicity and spontaneous release rates. [125I]Iododeoxyuridine was found unsuitable on account of its high toxicity and correspondingly high spontaneous release rate, and Na2 51CrO4 for its toxicity and low labelling efficiency. Of the two other radiolabels, [75Se]selenomethionine had the advantage over [3H]proline of higher labelling efficiency (especially in Ham's F10 medium), lower toxicity, and being a gamma-emitter. Furthermore, released 75Se was shown to be non-reutilisable and its retention by target cells provided an accurate measure of cell survival in an alloimmune system. Methods of calculating the results of pre-labelling cytotoxicity tests based on the total radioactivity in target cells at the beginning of the assay were found to be invalid.

Radiolabeled iododeoxyuridine: safety evaluation.

UNLABELLED: The emphasis of radiolabeled iododeoxyuridine (*IUdR) research at our institution to date has been to assess its safety as a potential therapeutic agent. Toward this goal, we have performed preclinical and clinical studies, using various routes of administration, to detect adverse changes in normal tissues in both humans and animals. As IUdR is rapidly dehalogenated by the liver, the intravenous route is unlikely to be successful in therapeutic efforts. We have therefore focused our attention on more "protected" routes: intra-arterial and intravesicular administration. METHODS: Studies were performed in farm pigs after multiple administrations of [125I]IUdR into the aorta, carotid artery and bladder. IUdR and metabolites were measured in venous blood samples at appropriate time intervals after administration, after which histologic examination of tissues was performed. Studies in human have been performed after intra-arterial administration of [123I]IUdR in patients with liver metastases and intravesicular administration in patients with bladder carcinoma, initially using [123I]IUdR and currently using both [123I]IUdR and [125I]IUdR. Blood samples for pharmacokinetics and metabolite analysis and tissue for autoradiography (when feasible) have been obtained. RESULTS: To date, no evidence of adverse effects on normal tissue or alteration of hematologic or metabolic indices have been seen in pigs or humans. When instilled in the bladder, there is little leakage of IUdR in the circulation. CONCLUSION: When [125I]IUdR is used as a therapeutic agent, we anticipate little or no effect on normal tissues.

An in vitro study of ophthalmic antiviral agent toxicity on rabbit corneal epithelium.

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.

Kinetics of micronuclei induced by 125IdU in cells of two lines.

The kinetics of the formation of cells carrying micronuclei (MN) after one doubling time (td) incorporation of 125I-iododeoxyuridine (125IdU) to Chinese hamster ovary (CHO) and rat anterior pituitary tumor (GC) cells was studied. Uptake of 125IdU by cells of both cell lines was linearly dependent on the concentration of extracellular 125I activity. The postlabeling time-dependent decrease in cellular activity of 125IdU was exponential in CHO cells and approximately linear in GC cells. The maximum yield of MN was seen during the second and third td after 125IdU incorporation. The frequency of cells with micronuclei increased monotonically with dose in the interval (1, 40) 125I decays cell-1td-1. The dose-response relationship could be fitted by straight lines with slopes of 1.0 (CHO) and 1.2 (GC) on the subinterval (1, 10) and of 0.6 or 0.5, respectively, for the subinterval (10, 40). Below one 125I decay cell-1td-1, the mean frequency of micronucleated binuclear cells was significantly lower than (CHO) or equal to (GC) the control. On average, one 125I decay/cell induced 0.95 +/- 0.5% (CHO) or 1.0 +/- 0.5% (GC) of micronucleated binuclear cells.

Targets for radiation-induced cell death: when DNA damage doesn't kill.

Chinese hamster ovary (CHO) K1 and radiosensitive CHO irs-20 cells were synchronized in S phase and labeled for 10 min with 5-[(125)I]-iodo-2'-deoxyuridine ((125)IdU). The cells were washed, incubated in fresh medium for 1 h for incorporation of the intracellular radionucleotides into DNA, and then frozen (-80 degrees C) for accumulation of (125)I decays. At intervals after freezing, when the cells had accumulated the desired number of decays, aliquots of the frozen cells were thawed and plated to determine survival. The survival curves for K1 and irs-20 cells were similar from 100% to 30% survival. At higher (125)I doses (more decays/cell), the survival of K1 cells continued to decline exponentially, but the survival of X-ray-sensitive irs-20 cells remained at approximately 30% even after the cells had accumulated 1265 decays/cell. The results contradict the notion that increased DNA damage inevitably causes increased cell death. To account for these findings, we propose a model that postulates the existence of a second radiation target. According to this model, radiation damage to DNA may be necessary to induce cell death, but DNA damage alone is not sufficient to kill cells. We infer from the survival response of irs-20 cells that damage to a second (non-DNA) structure is involved in cell death, and that this structure directly affects the repair of DNA and cell survival.

Proflavine photodynamic viral inactivation in herpes simplex keratitis.

We used the photodynamic inactivation technique with proflavine as the photoactive dye to treat herpetic epithelial keratitis in a preliminary study of patients who had idoxuridine toxicity or resistance. A comparative study with idoxuridine in treating dendritic ulcerations of the cornea showed a good therapeutic effect. But the investigation was suspended when adverse reactions, consisting of a generalized epithelial keratitis and an anterior uveitis, possibly of phototoxic origin, developed in a few patients receiving treatment. The ulcers treated by photodynamic inactivation apparently healed by a process of "debridement" followed by subsequent re-epithelialization.

Absence of delayed chromosomal instability in a normal human fibroblast cell line after 125I iododeoxyuridine.

PURPOSE: To seek the delayed appearance of chromosomal abnormalities in human fibroblasts exposed to the Auger electron emitter 125I. MATERIALS AND METHODS: Normal untransformed human fibroblasts, HF19, were exposed to a concentration of [I125]IUdR, which allowed the survival of 37% of clonogens. Chromosomal analysis using both conventional Giemsa and fluorescence in situ hybridization (FISH) was undertaken on non-clonal bulk cultures from 2 to 39 days after treatment. RESULTS: The data show a declining level of unstable aberrations in the progeny of HF19 fibroblasts exposed to [I125]IUdR, eventually reaching control levels. CONCLUSIONS: The results provide evidence that [125I]IUdR does not induce ongoing chromosomal instability in long-term culture, and gives further support to the use of Auger-electron emitting radionuclides in the treatment and diagnosis of tumours.

Chemical modification of 5-[125I]iodo-2'-deoxyuridine toxicity in mammalian cells in vitro.

PURPOSE: To address the cytotoxic effects of DNA-incorporated (125)I in Chinese hamster V79 lung fibroblasts under various scavenging conditions. METHODS: The toxic effects of DNA-incorporated 5-[(125)I]iodo-2'-deoxyuridine ((125)IdUrd) were assessed by the colony-forming assay with cells incubated in medium containing serum and/or dimethyl sulphoxide (DMSO). Experiments were carried out at 0.3 or -135 degrees C. RESULTS: When (125)I decays were accumulated at 0.3 degrees C in 10% serum 0, 5 or 10% DMSO, no radioprotection was afforded by 5% DMSO, while the dose modification factor (DMF) for 10% DMSO was 2.0. For cells accumulating decays at 135 degrees C in the presence of 5 or 10% serum, DMSO was radioprotective (DMF= 1.8-1.9). D(0) obtained at each serum concentration correlated strongly (R=0.999) with the scavenging capacity of DMSO. Under these experimental conditions, 10% serum is approximately 3.6 times more protective than 5% serum. CONCLUSIONS: The contribution of indirect mechanisms to the toxicity of (125)I decaying within mammalian cell nuclear DNA can be demonstrated not only with DMSO, but also with the hydroxy radical scavengers present in serum.

Anti-recombinogenic and convertible co-mutagenic effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted pyrimidine nucleoside analogs in S. cerevisiae MP1.

In experiments using yeast, without addition of an external metabolic activation system, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was co-mutagenic and showed an insignificant anti-recombinogenic effect in combination with triethylene melamine (TEM). In the presence of activating S9-mix, the anti-recombinogenicity and co-mutagenicity could clearly be seen. At higher concentrations the co-mutagenic effect was converted into anti-mutagenicity. The other three 5-substituted pyrimidine nucleoside analogs were tested only in the presence of activating S9-mix and showed similar effects. As TEM is a direct alkylating agent that is inactivated by liver microsomes, the higher activity in presence of S9-mix can be interpreted as resulting from metabolic activation of the 5-substituted pyrimidine nucleoside analogs. In previous experiments using yeast bacteria, Drosophila or mice, tumor promoters were co-recombinogenic/anti-mutagenic, and co-carcinogens were co-mutagenic/anti-recombinogenic. Thus, there is not only an operational difference between tumor promoters and co-carcinogens but a real difference in respect to their genetic effectiveness. As up to now only co-carcinogens have shown co-mutagenic and anti-recombinogenic effects, it is perhaps possible that, within a certain concentration range, 5-substituted pyrimidine nucleoside analogs may have co-carcinogenic activity in carcinogenicity tests. At higher concentrations the co-carcinogenic effect may be converted into an anti-carcinogenic one.

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting.

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 microg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28-79%) and to a lower extend at 1q12 (8-21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes.