Early and Late Effects of Difluorodeoxycytidine on Murine Cell Radiosensitivity In Vivo

Abstract

Background/Aim

Difluorodeoxycytidine (dFdC) has been reported to increase radiosensitivity, although its mechanism of action is unknown. The objective of this study was to determine the early and late effects of dFdC on mouse cell radiosensitivity in vivo.

Materials and Methods

The early effects of dFdC on bone marrow cell radiosensitivity were evaluated a few minutes after dFdC treatment using single-cell gel electrophoresis. Four groups of mice were set up: non-treated, dFdC-treated, radiation-treated, and dFdC plus radiation-treated. To evaluate the late effects of dFdC, the kinetics of micronucleus production and the inhibition of proliferation were measured in mouse normoblasts in vivo.

Results

The early radiosensitization index was 1.7 and correlated with the proportion of severely damaged cells, likely in S phase. Late effects of dFdC were additive with radiation in both micronucleus induction and cytotoxicity. The pattern and duration of micronucleus formation suggest a dependency on dFdC incorporation into DNA. Although cytotoxicity increased over time, it did not influence the radiosensitization index.

Conclusion

dFdC enhances early radiosensitivity in bone marrow cells, likely by inhibiting DNA synthesis and generating reactive oxygen species (ROS)-induced DNA breaks. Late genotoxic effects were additive and kinetically linked to dFdC incorporation. Both dFdC alone and in combination with radiation exerted prolonged cytotoxic effects on normoblast precursors, with slightly greater toxicity observed in the combination group.

Introduction

Difluorodeoxycytidine (dFdC) is a cytidine-derived nucleotide that has important antineoplastic effects on various types of cancer (1-3). These effects are probably due to the inhibition of ribonucleotide reductase activity, which reduces nucleotide pools and promotes dFdC incorporation (4), and the inhibition of thymidylate synthase, which causes dTMP reduction (5). dFdC is phosphorylated to dFdC triphosphate and is incorporated into DNA in vitro; however, DNA synthesis is interrupted one nucleotide after the incorporation of dFdC triphosphate, and DNA polymerase epsilon is unable to remove the incorporated nucleotide (6). dFdC has also been reported to inhibit topoisomerase in vitro (7) and induce the production of reactive oxygen species (ROS) (8,9).

dFdC has been reported to be a radiosensitizer (10), and in vitro assays have shown that its action depends on whether it is administered before radiation therapy (11). It has been suggested that the reduction in nucleotide pool caused the inhibition of ribonucleotide reductase activity (4) and the accumulation of cells in the S phase due to the inhibition of DNA synthesis (6) and this is the mechanism through which dFdC increases radiosensitivity. Importantly, this hypothesis explains an additive but not necessarily a synergistic effect.

These previous studies were conducted in vitro; however, the in vivo effects of dFdC may not be similar because of differences in the context, concentration and exposure time. Because biological systems are highly dynamic, monitoring over time is important for determining their action.

The objectives of the present study were to determine the early in vivo effects of dFdC on radiosensitivity and DNA damage using single-cell gel electrophoresis in mouse leukocytes and bone marrow cells in vivo, as well as to determine the long-term radiosensitization effects by evaluating the kinetics of micronucleus induction and the inhibition of proliferation in mouse normoblasts in vivo.

Materials and Methods

Animals. Male BALB/c mice at two months of age and weighing 30 g were maintained at 22-24˚C with a 12 h light/dark cycle and had access to laboratory mouse pellets (LabDiet, PMI Nutrition International LLC, Arden Hills, MN, USA) and water ad libitum. The animals were treated and maintained in accordance with the Official Mexican Guidelines (Norma Oficial Mexicana NOM-062-ZOO-1999). Protocols were approved by the CICUAL (Internal Committee for the Care and Use of Laboratory Animals) from the Instituto Nacional de Investigaciones Nucleares (ININ).

Chemicals. Agarose and low melting point (LMP) agarose were obtained from Gibco BRL (Gaithersburg, MD, USA); dFdC, tris, sodium chloride, N-lauroylsarcosine, dimethyl sulfoxide (DMSO), potassium chloride, and ethidium bromide (EtBr) were obtained from Sigma Chemicals (St. Louis, MO, USA); and methanol, EDTA, and sodium hydroxide were purchased from Merck (Darmstadt, Germany).

Determination of early DNA damage induction by dFdC in leukocytes in vivo. Five mice were intraperitoneally administered 10 µmol/kg of dFdC in an aqueous solution. Peripheral blood samples of 4 µl were collected from the tail at 0, 2, 5, 10, 15, 20, and 30 min after administration. The number of cells with a tail (comets) were scored.

Determination of dFdC-induced early DNA damage and repair in bone marrow cells in vivo. Three groups of five mice were intraperitoneally administered 10 µmoles/kg of dFdC in an aqueous solution. One group was sacrificed immediately, and the other two groups were sacrificed 60 and 120 min after the treatment. An untreated control group was also included. DNA damage in the cells was determined via single-cell gel electrophoresis, and the number of cells with a tail migration greater than 20 µm was quantified.

Evaluation of the early radiosensitizing effect of dFdC on bone marrow cells in vivo. The following four groups of mice were set up:

i. Control group, in which bone marrow cells were collected from untreated mice;

ii. dFdC group, in which dFdC was administered by intraperitoneal injection at a dose of 10 µmol/kg, and bone marrow cells were collected after 8 min;

iii. Irradiated group, in which the mice were individually exposed to 0.5 Gy of gamma radiation from a 60Co source (Gammacel, Atomic Energy of Canada Limited, Ottawa, Canada); and bone marrow cells were collected immediately afterward;

iv. Experimental group (dFdC+radiation), in which dFdC was administered by intraperitoneal injection at a dose of 10 µmol/kg, followed by exposure of the mice to 0.5 Gy of gamma radiation 8 min later and then collection of the bone marrow cells immediately afterward.

Evaluation of the long-term radiosensitizing effect of dFdC on mouse normoblasts in vivo. Three groups of mice (5 mice per group) were determined and treated as follows:

i. dFdC group, in which dFdC was administered by intraperitoneal injection at a dose of 47 µmoles dFdC/kg of body weight at the start of the experiment;

ii. Irradiated group, in which the mice were individually exposed to 0.5 Gy of gamma radiation from a 60Co source (Gammacel);

iii. Experimental group (dFdC+radiation), in which dFdC was administered by intraperitoneal injection at a dose of 47 µmol dFdC/kg of body weight at the start of the experiment, followed by exposure to 0.5 Gy of gamma radiation in a 60Co source (Gammacel) after 8 h. Peripheral blood samples were collected from the tail before treatment and every 8 h after treatment for up to 72 h.

Single-cell gel electrophoresis assay. This assay also known as comet assay permitted us to measure DNA damage in individual cells. During electrophoresis damaged DNA migrates away from the nucleoid forming a tail that resembles a comet. This assay allowed the frequency of damaged cells (comets) to be determined and the degree of damage to be established by measuring the tail migration.

A femur was dissected from each mouse and flushed with 1 ml of Hanks' solution to collect the bone marrow cells in a centrifuge tube on ice.

A previously reported procedure (12) was utilized, with some modifications (13). The samples were processed under low light. Fifteen microliters of each sample was centrifuged at 698×g for 10 min at 5˚C. The supernatant was discarded, and the pellet was resuspended in 100 μl of 0.5% low melting point agarose. This mixture was placed on a slide containing a layer of 0.75% normal melting point agarose and covered with a coverslip.

The slides were incubated at 4˚C for 5 min. Once the agarose solidified, the coverslip was removed by sliding, and another 100 μl layer of low melting point agarose was applied and covered with a coverslip, followed by refrigeration for an additional 5 min. The coverslips were then removed, and the slides were immersed in lysis solution at 4˚C for at least 1 h. The slides were then carefully transferred to an electrophoresis chamber and immersed in electrophoresis buffer (pH <13) at 4˚C for 40 min. Electrophoresis was performed for 40 min at 23 volts and 300 milliamps. Finally, the slides were rinsed with Tris-HCl buffer (pH 7.5) and dehydrated with methanol for subsequent analysis.

Staining was performed with 70 μl of ethidium bromide at a concentration of 20 μg/ml. The samples were analyzed with the Comet Assay IV Version 4.2 program (Perceptive Instruments, Haverhill, Suffolk, UK). DNA damage was measured in terms of tail migration. Fifty randomly selected cells were scored along the exposed zones on two slides, and 100 cells per mouse were imaged using a fluorescence microscope with an excitation filter of 546 nm, a barrier filter of 590 nm and a 25× objective.

Sample processing and analysis of micronucleated poly-chromatic erythrocyte (MN-PCE) induction kinetics and cytotoxicity kinetics (PCE frequency). Samples were collected from each mouse before treatment and every 8 h thereafter for up to 72 h. Samples (drop of blood) were collected from the tip of the tail through a small incision. The drop was then placed in a drop of fetal calf serum, and a smear was made and stained with May-Grunwald-Giemsa stain (14). MN-PCE counts were performed on 2,000 PCEs under a light microscope at 1,000X magnification. The proportion of PCEs in 2,000 erythrocytes was also determined as an index of cytotoxicity. The micronuclei that met the following criteria were counted under the microscope: round, diameter of approximately 1/20 to 1/5 of the diameter of the erythrocyte, and stained deep purple.

Results

Early DNA damage induction by dFdC in leukocytes in vivo. Figure 1 shows the early comet induction curve at different times from 0 to 30 min. Significant early induction was observed starting at 2 min, peaking at approximately 8 min, rapidly decreasing until 15 min, and decreasing more slowly until 30 min. Because the induction of DNA damage occurs during short durations in nondividing cells, it may not depend on altered DNA synthesis or on the inhibition of ribonucleotide reductase or thymidylate synthase.

Figure 1.

Early induction of comets in mouse leukocytes in vivo. The means±standard errors are presented, and asterisks indicate a significant difference from baseline. The Student's t test was used, and p<0.05 was considered to indicate statistical significance.

Early dFdC-induced DNA damage and repair in bone marrow cells in vivo. Figure 2 shows induction as a percentage of comets. The untreated control had 5% comets, but in cells obtained immediately after dFdC treatment, the proportion of cells with comets increased to 18%. This increase subsequently decreased to 15% at 60 min and to 8% at 120 min, which was not significant with respect to the control, suggesting that DNA damage was repairable and more rapid after 60 min.

Figure 2.

Induction of comets in bone marrow cells. The bars indicate the means±standard errors. Asterisks represent a significant difference compared with the untreated control. The Student's t test was used, and p<0.05 was considered to indicate statistical significance.

Early radiosensitizing effect of dFdC on bone marrow cells in vivo. Table I shows the tail migration results for the different treatments. Compared with the control group, all the groups presented highly significant differences. The tail migration in the group treated with dFdC and radiation (combined treatment group) was significantly greater than that in the groups treated with dFdC or radiation alone. The radiosensitization index, which was calculated in increments relative to the control value [(dFdC+radiation)-dFdC/(radiation)], was 1.7.

Table I. Effect of dFdC, radiation or combined treatment on tail migration in mouse bone marrow cells in vivo.

dFdC: Difluorodeoxycytidine; Rad: radiation. *Significant difference vs. control, p<0.002 (Student’s t test).

Figure 3 shows the tail migration curves of bone marrow cells in increasing order for all the mice, with points representing each cell and the line representing the average. The average curves for the control mice and the mice treated with dFdC, radiation, or the combination of dFdC and radiation are shown. Treatments with dFdC, radiation, and the combination of dFdC and radiation clearly increased the number of cells with more damage in terms of tail migration, although important variability was observed. This variability was normal for in vivo experiments, but it might be increased owing to variable proportions of bone marrow cells undergoing DNA synthesis.

Figure 3.

Tail migration curves for cells from control mice and those treated with dFdC, radiation, or the combination of dFdC plus radiation. The curves are arranged in progressive order. The thick line represents the average of the curves for all the animals.

To simplify the comparison between the progressive order curves of cell tail migrations from mice with different treatments, the averages of the curves for the different treatments were drawn (Figure 4). The control curve for leukocytes was included to demonstrate that in untreated bone marrow, there were more breaks in the DNA due to the large number of cells undergoing DNA synthesis.

Figure 4.

Average tail migration curves for the controls and different treatments. The control curve for leukocytes is included. The horizontal lines at 25, 50, 75, and 100 μm allow comparison of the response to the different treatments.

The curve for the combined treatment consisted of two components. The first component was the number of cells with tails up to 100 µm, which showed a linear increase and was almost proportional to the control curve, that is, to the DNA breaks produced during DNA synthesis, unlike the curves for the dFdC and radiation treatments, which showed a preferential increase in short tails. The second component of the combined treatment curve was the number of cells with tails longer than 100 µm, which showed an exponential increase. This exponential increase could be due mainly to cells in the DNA synthesis stage, which represented approximately 35% of the total cells. The amount of damage to these cells was likely incompatible with the viability.

Figure 4 shows that the sensitization in the combined treatment group was clearly associated with greater tails, which was reflected by the calculated radiosensitization indices for different migration levels (Table II). For a tail length of 25 µm, no increase in the radiosensitization index was observed. At 50 µm, an additive effect of dFdC+radiation was observed, but at 75 µm and 100 µm, the sensitization indices increased to 2.2 and 12.3, respectively.

Table II. Radiosensitization index (RI) calculated from different size tail migrations (from Figure 4).

Rad: Radiation; dFdC: difluorodeoxycytidine.

Long-term radiosensitizing effect of dFdC in mouse normoblasts in vivo. Figure 5 shows the average curves of MN-PCE induction by dFdC, radiation, and the combined treatment with dFdC+radiation. The area beneath the curve (ABC) of the response indicated an additive effect because the sum of the ABCs for MN-PCE induction by dFdC and radiation was nearly equal to that of the combined treatment. The response to individual treatment with dFdC or radiation alone is presented with average curves with a maximum of 33 MN-PCE at approximately 30 h. However, the induction period was longer in the case of radiation, from 12 to 52 h, which is 40 h, than that of dFdC from 20 to 44 h, which is 24 h. The peak of the curve of the combined response was significantly greater with a maximum at 46 h and lasted from 16 to 52 h, which is only 36 h. Dividing the peak height by the base width indicated that dFdC and the combined treatment caused a sharper peak, unlike the peak caused only by radiation, which was wider. These findings suggested that the induction of MN-PCE in the combined treatment group was determined by the effect of dFdC, perhaps by its incorporation into DNA. The peak in the combined group was delayed by approximately 6 h, even though irradiation occurred 8 h later. Unlike what might be expected, only one peak was formed, not two; one from the dFdC treatment and another from the radiation exposure 8 h later. These findings suggested a different interaction of radiation with DNA pretreated with dFdC than with radiation alone but with no impact on the amount of damage.

Figure 5.

Micronucleated polychromatic erythrocyte (MN-PCE) frequency as a function of time in mice treated with dFdC, radiation, or both. In the combined group, radiation exposure occurred 8 h after dFdC treatment. Asterisks indicate a significant difference (Student's t test, with p<0.05 indicating significance) compared with time zero before treatment, which is considered the control for each mouse. ABC: Area beneath the curve.

Figure 6 shows the cytotoxicity curves in terms of the percentage reduction in the PCE determined in 2,000 erythrocytes. Compared with treatment with dFdC alone, radiation induced an increase in the ABC of approximately 20% in the curve of dFdC-pretreated cells. dFdC produced cytotoxicity for a long period, from 16 h to 64 h, with a maximum of 80% at 40 h. Because the increase in the ABC of the curve from the combined group was consistent at all time points with respect to the group treated with dFdC alone, the comparison at each time point by paired t test indicated that the difference was highly significant (p<0.002). Unlike genotoxicity curves, which increase at specific time points, radiation curves of cells pretreated with dFdC exhibited increased cytotoxicity over time. These findings suggested that genotoxicity determined by micronucleus production was not related to cytotoxicity. Considering the ABC as an index of total cytotoxicity, the results indicated that the combination of dFdC and radiation had an effect slightly less than the sum of the two treatments separately.

Figure 6.

A reduction in the polychromatic erythrocyte (PCE) frequency is an index of cytotoxicity. The points represent the means, and the bars represent standard errors. Asterisks indicate a significant difference (paired Student's t test, p<0.05) with respect to the values at time zero considered the control for each mouse. ABC: Area beneath the curve.

Discussion

Induction of DNA damage by dFdC in leukocytes. In the present study, a comet assay revealed early induction of DNA breaks in mouse peripheral blood leukocytes within a few minutes after dFdC administration in vivo. Considering the rapid response and that the leukocytes were not dividing, the inhibition of the nucleotide-metabolizing enzymes ribonucleotide reductase and thymidylate synthetase (4,5), as well as the inhibition of DNA synthesis and topoisomerase (6,7), were ruled out.

dFdC reached its maximum effect at 8 min after administration, as evidenced by its ability to produce DNA breaks. A plausible explanation for the early DNA damage, occurring independently of metabolism and DNA synthesis, is the generation of reactive oxygen species (ROS) induced by dFdC (8,9) through an as-yet unidentified mechanism, leading to the formation of early DNA strand breaks.

Repair of early DNA damage caused by dFdC in bone marrow cells. Considering that cancer cells are continuously dividing, establishing an early response to dFdC in dividing cells is important. The present results revealed early DNA damage caused by dFdC and its repair in mouse bone marrow cells in vivo. dFdC immediately produced almost three times the number of breaks present in untreated bone marrow cells. The early DNA damage caused by dFdC gradually decreased over time; at 120 min, the amount of damage, although still greater, was not significantly different from that of the untreated control. This finding indicated that the DNA damage immediately produced by dFdC could be repaired.

In addition to the possible damage caused by ROS, dFdC may act on sites undergoing DNA synthesis in dividing cells, inhibiting the progress of synthesis early (15). Therefore, damage is less likely to be a consequence of the effect of dFdC on nucleotide metabolism (4,5).

Moreover, dFdC blocks DNA synthesis but does not form new breaks. It has been reported that the inhibition of DNA synthesis by dFdC is not reversible, at least not in the short term (6). In the present study, the DNA breaks were repairable after one hour, suggesting that these breaks were not generated via pathways involved in the inhibition of DNA synthesis.

An alternative mechanism is the production of ROS by dFdC, which has been previously established because dexamethasone sensitizes cancer stem cells to dFdC by increasing ROS production through the reduction of NRF2, a key regulator of antioxidant responses, and the effect can be reversed by the N-acetyl-L-cysteine free radical scavenger (16).

Early effect of dFdC on bone marrow cell radiosensitivity. By measuring mammary tumor survival in mice, researchers reported in 1992 that dFdC induces radiosensitization in vitro (10). Since this report, various studies have investigated the repair processes involved in radiosensitization.

Although the involvement of the nonhomologous end-joining pathway in dFdC radiosensitization has been ruled out (17), it remains unknown whether dFdC induces radiosensitization in base excision repair-deficient cells. However, homologous recombination repair-deficient cells do not exhibit dFdC-mediated radiosensitization, suggesting the involvement of homologous recombination (18). RAD51-dependent knockdown of homologous recombination has been demonstrated to be essential in dFdC-induced radiosensitization (19).

There is also evidence that mismatch repair deficiency further increases the radiosensitivity induced by dFdC (20). Only mismatch repair-deficient cells showed an increase in nucleotide misincorporation (2- to 3-fold, p>0.01) after radiosensitization with different concentrations of dFdC and 5.0 Gy of radiation, and this increase persisted for at least 96 h (21).

Considering that radiation was administered only 8 min after dFdC injection in the present study, it is unlikely that repair processes were involved in dFdC-induced radiosensitization. The findings of this study indicate that both dFdC and radiation induce DNA strand breaks, as evidenced by the presence of short tail migrations in the comet assay. However, the mechanism by which the DNA breaks induced by dFdC are further fragmented by radiation to produce longer DNA tails remains unclear. In addition, the incorporation of dFdC at DNA synthesis sites inhibits this process (6), which would explain the persistence of breaks but not the induction of more breaks in the short term.

In the present study, radiation had a synergistic effect on cells pretreated with dFdC for 8 min, resulting in a radiosensitization index of 1.7. These findings suggested that the combination therapy had early synergistic effects. An alternative explanation is the generation of ROS by dFdC (8,9), which could cause DNA breaks. A previous study reported that dexamethasone decreases the expression of NRF2, a known regulator of the cellular response to oxidative stress, thus controlling the amount of intracellular ROS. The combination of dFdC and dexamethasone causes a substantial increase in ROS levels; although dexamethasone does not directly produce ROS, dFdC-induced production of ROS is more evident when NRF2 expression is decreased by dexamethasone (16). The present study suggests, the amount of ROS produced by dFdC and radiation likely overcame the antioxidant response. Two early effects of dFdC seem to participate in radiosensitization: the induction of DNA breaks by ROS and the inhibition of DNA synthesis (6). This hypothesis is in accordance with data showing that the greatest effect on radiosensitivity was observed after treatment with dFdC, which resulted in the highest percentage of S-phase cells (15). Although it has been reported that the fragmentation caused by radiation in cells pretreated with dFdC is reversed within 1 h of incubation to allow for repair (22), fragmentation could be observed long after dFdC treatment in the present study.

Late radiosensitizing effect of dFdC in mouse normoblasts in vivo. The in vivo assay of micronucleus induction kinetics in polychromatic erythrocytes (as an index of DNA break induction in normoblasts) and the kinetics of the polychromatic erythrocyte frequency (as an indicator of cytotoxicity in normoblasts) allowed the genotoxic and cytotoxic effects of different agents to be monitored simultaneously (23). This in vivo assay has the following unique characteristics: i) it allows detection of the genotoxic and cytotoxic effects in populations of the same cell lineage in the same blood sample; ii) it is an in vivo model; iii) it is used in cells that are undergoing continuous proliferation, a characteristic that they share with neoplastic cells; iv) it allows kinetic studies to be performed by periodically taking small samples of blood from the tail; v) its procedure is noninvasive; vi) each animal serves as its own control; and vii) it allows dosimetric approximations for humans, although appropriate caution should be taken (24).

In the present study, the long-term radiosensitizing effect of dFdC was explored using the micronucleus induction in vivo. The doses of dFdC and radiation used in the present study demonstrated only an additive effect in terms of both genotoxicity and cytotoxicity. The genotoxicity findings suggested that a different interaction occurred when DNA pretreated with dFdC was exposed to radiation, but the interaction did not affect the amount of damage. Because radiation exposure occurred 8 h after dFdC administration, which implies that the DNA was modified by dFdC, the most viable alternative would be the incorporation of dFdC into DNA.

Transient siRNA knockdown of deoxycytidine kinase (dCK) has been reported to significantly reduce the radiosensitization of cells by dFdC. These findings imply that radiosensitization depends on the incorporation of dFdC into DNA, which inhibits DNA synthesis in the first step (6). There is evidence that early breaks in DNA generated by dFdC (Figure 2), as well as by the combination of dFdC and radiation, are reversed after one hour (22). Although homologous recombination (19) and mismatch repair (21) have been implicated in this phenomenon, no evidence has been reported regarding the fate of dFdC incorporated into DNA, which initially blocks DNA synthesis. It is unclear whether the incorporated dFdC is a lesion that is tolerated and synthesis is restarted or if it is terminated. If dFdC is incorporated, the incorporation opens the possibility that free radicals formed by radiation react mainly with the flour of dFdC, causing a new kind of lesion, as has been reported for bromine incorporated by bromodeoxyuridine (25).

In terms of cytotoxicity, the kinetics of dFdC and combination treatment were similar. Cytotoxicity began early and lasted up to 64 h, with a maximum of 80% at 40 h, in the dFdC-treated group, and between 30 and 50 h in the combined treatment group. In the combined treatment group, there was a small but significant, consistent increase in cytotoxicity throughout the entire kinetics period in the dFdC-treated group. There was a 20% increase in the ABC of the combination treatment compared with that of the dFdC alone treatment, but this increase was not synergistic because the ABC of the kinetics of the cytotoxicity in cells treated with radiation was high. Notably, the radiation kinetics were significant only at 40 h compared with the baseline value. This finding indicated that radiation of cells pretreated with dFdC resulted in an increase in cytotoxicity of approximately 10% throughout the entire posttreatment period. This response suggested that dFdC and the combined treatment had a persistent cytotoxic effect on precursor normoblast cells in the bone marrow and that this effect was slightly greater with the combined treatment.

Comparison of the kinetics of genotoxicity and cytotoxicity caused by dFdC revealed that these events did not coincide in time; cytotoxicity began early and lasted much longer than the genotoxic effect, which occurred at a specific, discrete time. As cytotoxicity in the combined treatment increased over time, following kinetics similar to those caused by dFdC, the cytotoxicity was dependent on the effect of dFdC and not entirely dependent on the process of inducing DNA breaks that cause micronuclei. The late cytotoxic effect may be explained by the effect of dFdC on normoblast precursor cells.

Conclusion

The present study demonstrates that dFdC induces DNA breaks in leukocytes a few min after treatment, probably through ROS production. The early DNA damage caused by dFdC can be repaired within the first hour posttreatment. dFdC has a radiosensitizing effect on bone marrow cells in the first few min after treatment, probably caused by blocking DNA synthesis and the generation of ROS-induced DNA breaks.

The late radiosensitizing effect of dFdC pretreatment on the induction of micronuclei by radiation indicates an additive effect, although it is kinetically dependent on the presence of dFdC. The late cytotoxic effect indicates that dFdC and the combined treatment cause a persistent cytotoxic effect in the precursor normoblast cells in the bone marrow, but it is slightly greater with the combined treatment.

In normoblasts, the kinetics of cytotoxicity caused by dFdC alone or the combination of dFdC with radiation do not temporally coincide with the kinetics of genotoxicity.

Conflicts of Interest

The Authors have no conflicts of interest to declare in relation to this study.

Authors’ Contributions

PMR and VCV contributed to the conception and design of the study. PMR, VCV, TVK JQV and SLP contributed to the development of the experiments, acquisition of the data, and analysis and interpretation of the data. PMR, VCV, TVK, JQV and SLP drafted the article and revised it critically for important intellectual content. All the Authors approved the submitted version of the manuscript.

Acknowledgements

The Authors would like to thank Angel Reyes Pozos and Miguel Ángel Torres for their excellent technical assistance. The Authors would also like to thank Dr. Pedro R. González for assisting with the radiation dosimetry.

Funding

Funds were provided by Project BI-001 from the Instituto Nacional de Investigaciones Nucleares (ININ). Support for part of this work was given to Dr. Virginia Cruz-Vallejo by the Fondo para la Investigación Científica y Desarrollo Tecnológico del Estado de México (No. FICDTEM-2023-04).

Artificial Intelligence (AI) Disclosure

No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.