Radiosensitization of murine normoblasts in vivo by bromodeoxyuridine to the genotoxicity and cytotoxicity of the bone-seeking radiopharmaceutical 153Sm-EDTMP.

Abstract To establish a basis for a possible strategy for bone marrow ablation or therapy, we examined the effect of bromodeoxyuridine (BrdU) incorporation into DNA on the genotoxic and cytotoxic effects of samarium-153 ethylenediaminetetramethylene phosphonate ((153)Sm-EDTMP) in normoblasts in vivo. Cytotoxicity and genotoxicity were established by time-response curves of polychromatic erythrocyte (PCE) and micronucleated polychromatic erythrocyte (MN-PCE) frequencies, respectively, in mouse peripheral blood samples. The group treated with (153)Sm-EDTMP showed a clear induction of MN-PCEs; however, the group treated with BrdU plus (153)Sm-EDTMP paradoxically showed only a slight increase with respect to untreated controls. Treatment with (53)Sm-EDTMP caused a small reduction in PCE frequency, but exposure to BrdU or to BrdU plus (53)Sm-EDTMP reduced the PCE frequency significantly from 32 h to the end of the experiment. The PCE frequencies in the BrdU plus (53)Sm-EDTMP group were significantly lower than in the BrdU control group at the final time and were much lower than the group treated with only (53)Sm-EDTMP, which returned to basal values. The results suggest the radioinduction of a lethal lesion in BrdU-substituted DNA that cannot be repaired easily and does not permit cell division and micronucleus formation.

Effect of O6-chloroethylguanine DNA lesions on the kinetics and mechanism of micronucleus induction in vivo.

The aim of this work was to determine the kinetics of micronucleus production because of an increase in O(6)-chloroethyl guanine (O6-ChlEt-G) DNA lesions in murine bone marrow cells in vivo. We increased the frequency of O6-ChlEt-G lesions by pretreatment with an inhibitor of O(6)-methylguanine-DNA methyltransferase (MGMT), O(6)-benzylguanine (O6BG), and subsequent treatment with bis-chloroethylnitrosourea (BCNU). The kinetics of micronucleated-polychromatic erythrocyte (MN-PCE) induction was established by scoring the frequency of MN-PCEs per 2000 PCEs in peripheral blood at 8-hr intervals from immediately prior to treatment to 72-hr post-treatment. We examined groups of five mice treated with (i) dimethylsulfoxide (DMSO), (ii) O6BG in DMSO, (iii) BCNU, or (iv) O6BG in DMSO plus BCNU. The data indicate that O6BG pretreatment causes: (i) ían increase in MN-PCEs induced by BCNU, (ii) a delay in the time of maximal MN-PCE induction produced by the different BCNU doses, and (iii) an increase in cytotoxicity. These data confirm that O6-ChlEt-G is a lesion involved in DNA break induction and in the subsequent production of micronuclei, and also that these lesions seem to be stoichiometrically reduced by MGMT. These data also show that induction of MN-PCEs by BCNU is delayed by pretreatment with O6BG for more than 6 hr, perhaps due to the time required for repair of crosslinks derived from O6-ChlEt-G and/or for DNA duplication, which is required for adduct transformation into crosslinks.

Mechanisms of DNA breaks induction in vivo by 5-azacytidine: paths of micronucleus induction by azaC.

The aim of the present study was to correlate the time-response curves of micronucleated polychromatic erythrocyte (MN-PCE) induction by 5-azacytidine (azaC) with the possible processes involved in DNA break production; this is based on the results previously published by other authors. The MN-PCE induction at two different doses of azaC was determined by sampling blood from the tails of mice before the acute treatment and over nine periods of 8 h each afterwards. Both doses caused two peaks of MN-PCE induction, one at 32 h and another at 48 h, approximately; a shoulder was detected that remained high from 56 h up to the end of the study (72 h). These results suggest that azaC induced DNA breaks and subsequently MN (micronucleus) by three different mechanisms, and in agreement with data in the literature, these could be successively the following: (i) during excision of the large adduct comprising the DNA methyl transferase covalently linked to DNA; (ii) failure of recombination repair or mismatch repair; and (iii) persistent chromosome fragility in G-C rich sites due to DNA demethylation and chromatin decondensation.

Kinetics of micronucleus induction and cytotoxic activity of colchicine in murine erythroblast in vivo.

In previous studies, we inferred some pharmacokinetic and pharmacodynamic parameters of alkylating agents and antimetabolites by comparing their kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction with the one obtained after the exposure to gamma rays in peripheral blood of mice, assuming that radiation acts immediately because it does not require absorption and distribution in the organism. According to our earlier studies, the kinetics of MN-PCE induction depends mainly on the following: (i) the cytotoxic effects that in turn could affect the duration of cell division; (ii) the pharmacokinetics including the metabolic activation requirement; and (iii) the mechanism of MN induction. The aim of the present study was to analyze the kinetics of MN-PCE induction by an aneuploidogen that induces micronuclei by acting on the achromatic spindle. The kinetics of MN-PCE induction by colchicine, as well as the reduction in the PCE frequency over time was determined in peripheral blood of mice treated with different doses of the aneuploidogen. The genotoxic effect, established as the area beneath the curve (ABC) of MN-PCE versus time-response, indicates an almost directly proportional relationship with respect to dose. Similarly, the relationship between dose and cytotoxic effect determined as the ABC of PCE versus time was inversely proportional, suggesting a relationship between both endpoints and doses administered. However, the number of cells affected by these two phenomena indicates that cytotoxicity is not necessarily caused, or at least not only by genotoxicity. The analysis of the kinetics of MN-PCE induction after the treatment with non-cytotoxic dose of colchicine, indicates that the MN-PCE appear in the blood stream at almost the same time, as occurs after the exposure to gamma rays; in spite of the differences in the cell cycle stage in which they can cause micronucleus (MN). Perhaps the fact that cells are not synchronized does not permit one to observe some difference in the time they appear in the blood. These results suggest that colchicine acts rapidly after exposure. The elimination half-life of colchicine is 17h, suggesting that colchicne is disposable for long time. With high doses of colchicine the pharmacokinetic parameters increases substantially. These data imply that low doses of colchicine are slightly cytotoxic, and that under this circumstances colchicines arrives rapidly to hemopoyetic tissues and acts for several hours.

Relationship between the kinetics of micronuclei induction and the mechanism of chromosome break formation by methylnitrosourea in mice in vivo.

The kinetics of micronucleated polychromatic erythrocytes (MN-PCE) induction by methylnitrosourea (MNU) was determined in mice with the purpose of discerning whether or not the kinetics reflects the mechanism of chromosome break induction. A very long latency period (LP) was observed which is not compatible with an agent that does not require metabolic activation or incorporation to DNA for acting, but this is consistent with the mechanism demonstrated earlier that MNU causes chromosome breaks throughout the repair of mismatches induced by the alkylation of bases in a previous division. This is also supported by the presence of two rates of MN-PCE induction with respect to dose, which suggests that MN-PCE are induced by two mechanisms, an efficient one induced with the lower dose, and another less efficient one induced with higher doses. A similar behavior was observed in the curve of LP vs. dose, the lower dose causes 8 h of LP and higher doses increase LP but not proportionally to dose. The lower dose did not cause a reduction in the proportion of polychromatic erythrocytes, suggesting that this dose did not produce an important cytotoxic effect that could explain the long LP.

Pharmacokinetic parameters determined from the clastogenic activity of ethylnitrosourea and dimethylnitrosamine in mice in vivo.

The latency period (LP) and the time of effective activity (TEA) of ethylnitrosourea (ENU) and dimethylnitrosamine (DMN) were inferred by comparing their kinetics of micronucleated polychromatic erythrocytes (MN-PCE) formation with the kinetics induced by radiation. The results indicate that LP and TEA vary between ENU and DMN. For ENU, these parameters are very similar to radiation indicating a rapid distribution, reaction and elimination. DMN presents a very long LP which agrees with the requirement of mutagen activation. The kinetics of MN-PCE production caused by DMN showed two peaks; this could be due to the presence of two different metabolites, two types of lesions in DNA or two mechanisms of MN-PCE formation. These hypotheses do not exclude each other. The data presented here support the conclusion that the comparison of MN-PCE-formation kinetics induced by chemical agents with that caused by radiation permits one to estimate the LP and the TEA, and provide information on the possible mechanism of action of chemical mutagens.

Pharmacokinetic parameters of genotoxic activity inferred from the comparison of the kinetics of MN-PCE induced by chemical agents and ionizing radiation.

Some kinetic parameters of clastogenic activity of cyclophosphamide were inferred by means of the comparison of its kinetics of micronucleated polychromatic erythrocytes (MN-PCE) formation with the kinetics induced by radiation. The same reasoning was also applied to the kinetics obtained by treatment with mitomycin C (MMC), arabinocyl cytosine (Ara-C) and 6-mercaptopurine (6-MOP), based on previously reported data from the literature. The results indicate that the latency period (LP) and half-lives (HL) vary from one mutagen to another. For MMC, they are very similar to radiation indicating a rapid distribution and reaction. CP presents very long LP and HL which agree with the requirement of mutagen activation. Ara-C showed a very short LP which suggests a rapid activation and fast induction of damage in DNA. 6-MOP presented a very long LP which agreed with the requirement of its incorporation into DNA to cause micronucleus (MN). From the data obtained in the present work, it can be concluded that the comparison of the kinetics of MN-PCE formation induced by chemical agents with that obtained by the exposure to an acute dose of radiation permits one to estimate some parameters of the kinetics of clastogenic activity of chemical agents, like the LP and the HL. This seems to be valid for agents that act through the induction of DNA lesions; in the case of agents whose clastogenic activity is through other mechanisms, such as the inhibition or alteration of the process of duplication of the DNA, the kinetic parameters are not equivalent to the LP and HL; however, they could provide information on their possible mechanism of action.

No radioadaptive response to micronucleated polychromatic erythrocyte (MN-PCE) induction in murine peripheral blood in vivo.

The effect of conditioning pretreatment with 0.025 Gy of gamma rays on micronucleated polychromatic erythrocyte (MN-PCE) induction by 1.0 or 0.1 Gy of gamma rays was determined in murine peripheral blood. The adaptive and challenge doses as well as the timing of their administration were taken from a previously reported experiment [Farooqi and Kesavan (1992). Mutat Res 302:83-89]. The response was determined by the strategy of measuring the area below the curve (ABC) of MN-PCE induction vs. time. This strategy permits one to determine an index of total damage and to establish if conditioning exposure affects the timing of MN-PCE appearance in the blood stream, which in turn could cause an apparent difference in response between the conditioned and the unconditioned groups at specific times. The results indicate that low dose gamma ray pretreatment does not protect against MN-PCE induction by the challenge gamma ray dose, and that there was no change on the kinetics of MN-PCE appearance in peripheral blood.

Effect of chlorophyllin on gamma ray induced micronuclei in polychromatic erythrocytes of murine peripheral blood determined by the ABC strategy.

The effect of chlorophyllin on micronucleated polychromatic erythrocyte (MN-PCE) induction by gamma ray exposure in peripheral blood of mice was studied. The area beneath the curve (ABC) of MN-PCE frequency versus time was used as an index of total MN-PCE induction. The dose of 200 mg chlorophYllin per kg of body weight caused a slight, but not significant, reduction of the MN-PCE caused by 1.0 Gy exposure. This result indicates that chlorophyllin did not protect the cells against MN induction. In previous studies it was observed that the same chlorophyllin dose was able to protect 100% against sister chromatid exchange (SCE) induction by 1.0 gamma rays in both murine spermatogonia and bone marrow cells. These contradictory results indicate that chlorophyllin did not protect cells by scavenging free radicals, but by other mechanism, i.e. stimulating repair of lesions involved in SCE induction.

Fate of lesions that elicit sister chromatid exchanges (FLE-SCE) produced in DNA by alkylating agents in vivo.

A study was made on the effect of ethyl methanesulfonate (EMS) or dimethylnitrosamine (DMN) on the frequency of SCE occurring in the first, in the second or at the same locus in both divisions. This was done with a previously reported in vivo protocol which allowed us to determine the fate of lesions that elicit SCE (FLE-SCE) in two cell divisions after mutagen treatment. The results showed that EMS-induced DNA lesions that cause SCE are persistent, and that some of them were produced in the second-division or were generated from SCE non-inducing lesions. We inferred this by the analysis of the response in cell populations. DMN seems to induce SCE mainly during the second division, but by inhibiting DNA synthesis as was interpreted from the cell frequency distribution with respect to the number of SCE in first and in second cell division.

Persistence during G1 of gamma ray- or mitomycin C-induced lesions eliciting SCE in murine salivary gland cells in vivo.

The efficiency of mitomycin C or gamma rays to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation before S. The SCE frequencies induced by MMC in the first division (before BrdU incorporation) were significantly lower in the early G1 compared to the late G1, indicating some repair of SCE-inducing lesions. In the second division (after BrdU incorporation), there was no difference between SCE induced in early and late G1, indicating that MMC-induced lesions in such conditions are very persistent and not repairable during G1. The radio induced SCE frequency at early G1 was significantly lower than that observed in late G1, in cells irradiated after BrdU incorporation, suggesting that half of gamma ray-induced DNA lesions that elicit SCEs were repaired.

Induction of micronuclei by acute and chronic exposure in vivo to gamma rays in murine polychromatic erythrocytes.

The effect on micronuclei (MN) frequency of in vivo exposure to different dose rates of gamma rays in murine polychromatic erythrocytes (PCE) was studied. Groups of animals were irradiated with 1.0 Gy of gamma rays administered in 10, 100, 1000 or 10,000 min; the micronucleated polychromatic-erythrocytes (MN-PCE) frequency was scored in blood samples obtained from the tail of mice at various times before, during or after irradiation. The time-response curves for the 10, 100, and 1000 min exposure were similar; however, the two first curves showed a peak at 1800 min and the last at 2400 min after exposure. The curve obtained from the 10,000 min exposure showed a plateau for a long period during and after the exposure. However, the integration of the area under the curves indicates that the damage caused by the different radiation protocols was the same, suggesting that throughout time, the lesions were not repaired but rather diluted by cell division.

Comparison of sister chromatid exchange induction in murine germinal and somatic cells by gamma radiation exposure in vivo.

Sister chromatid exchange (SCE) induction by gamma rays was determined in spermatogonia irradiated before or after BrdU incorporation. Furthermore, the comparison of responses obtained in spermatogonia, bone marrow and salivary gland cells was carried out in the cells irradiated after BrdU incorporation, a condition which permits a higher SCE induction. Results indicate that gamma ray exposure of spermatogonia could induce a significant increase in SCE frequency with doses as low as 0.27 Gy, either before or after BrdU incorporation. However, the increase caused by radiation exposure after BrdU incorporation in spermatogonia was nearly three times lower than that obtained in both bone marrow and salivary gland cells. These data suggest that spermatogonia are either more efficient in repairing the gamma ray-induced lesions involved in SCE production or that these cells are less prone to the induction of such lesions.

In vivo fate of MMS-induced DNA lesions that elicit SCE.

A previously reported in vivo protocol, which uses three-way differential staining (TWD) of sister chromatids, allows the screening of mutagen-induced sister-chromatid exchange (SCE) in each of the two cell divisions after mutagen treatment and also those occurring at apparently the same locus in both divisions. In the present work the effect of methyl methanesulfonate (MMS) was studied by means of this protocol. The results showed that MMS-induced DNA lesions that cause SCE are persistent. Some lesions were induced in the second division, as was inferred from the analysis of the response in single cells. The data also indicate that bromodeoxyuridine reduces DNA sensitivity to SCE induction by MMS.

Fate of DNA lesions that elicit sister-chromatid exchanges.

Using 3-way differential staining (TWD) of sister chromatids, the fate of DNA lesions involved in sister-chromatid exchange (SCE) formation was determined in murine bone marrow cells in vivo, after treatment with either mitomycin C (MMC) or cyclophosphamide (CP). Both MMC (2.6 mg/kg b.w.) and CP (7 mg/kg b.w.) induced an SCE frequency near the expected in the 2 subsequent cell divisions, but the frequency of SCE occurring at the same locus in successive cell divisions was substantially lower than expected. The results are compared with previous data obtained after exposure to gamma-rays. A model of SCE induction is proposed.

Occurrence in vivo of sister chromatid exchanges at the same locus in successive cell divisions caused by nonrepairable lesions induced by gamma rays.

The capacity of lesions induced by gamma radiation to produce sister chromatid exchanges (SCE) in successive divisions in mouse bone marrow cells in vivo was evaluated using a protocol for the three-way differentiation of sister chromatids. Evidence was obtained that exposure to gamma radiation induces DNA lesions that result in the formation of SCE at the same locus in two successive cell divisions. The relevance of this observation with respect to DNA repair and mutagenesis is discussed.

Analysis of spontaneous sister-chromatid exchanges in vivo by three-way differentiation.

By applying an adaptation of the method of three-way differentiation to murine bone marrow cells in vivo, the basal frequency of sister-chromatid exchange (SCE) per cell was evaluated. An SCE frequency directly proportional to the estimated relative incorporation of 5-bromodeoxyuridine (BrdU) to the chromosomes was observed for the 3 consecutive cell cycles, implying that the majority, if not all, of the SCEs in vivo were produced by the incorporated BrdU. This conclusion was supported by the finding that in the first cycle of division, a very high frequency of cells without SCE was observed. From these data, a spontaneous frequency of SCE as low as 0.15 SCE/cell/cell cycle was inferred.

In vivo persistence of sister chromatid exchanges (SCE) induced by gamma rays in mouse bone marrow cells.

The sister chromatid exchange (SCE) frequencies induced in bone marrow cells by in vivo irradiation with gamma rays before or after bromodeoxyuridine (BrdUrd) incorporation were compared. The frequency of SCE at different postirradiation times was also measured in bone marrow cells in vivo, irradiated before BrdUrd incorporation. Increased sensitivity to SCE induction by radiation was found in cells after BrdUrd incorporation for one cycle when compared with cells irradiated before BrdUrd incorporation. The increased SCE frequency persisted for at least 72 hr after the initial irradiation, implying that the gamma ray-induced lesion(s) capable of eliciting an SCE are persistent and cannot be easily repaired.

Detection of SCE in rodent cells using the activated charcoal bromodeoxyuridine system.

An in vivo method of sister chromatid differentiation based on the intraperitoneal (ip) injection of bromodeoxyuridine (BrdUrd) previously adsorbed to activated charcoal is described, as well as the protocols for the use of this method in spermatogonial, salivary gland and bone marrow cells, will be presented. Results on sister chromatid exchange (SCE) induction obtained in bone marrow cells using well-known mutagens, especially cyclophosphamide, are reported, and a comparison is made with the results obtained by other authors using different methods of BrdUrd administration. The usefulness of this in vivo system for chemical mutagen detection and for chemical mutagen action on SCE induction is discussed.

Effect of BrdUrd and low doses of gamma radiation on sister chromatid exchange, chromosome breaks, and mitotic delay in mouse bone marrow cells in vivo.

We have measured, in mouse bone marrow cells in vivo, the ability of low doses of gamma radiation to induce sister chromatid exchanges (SCEs) in unifilarly 5-bromodeoxyuridine (BrdUrd)-substituted DNA. Also examined was the effect of BrdUrd incorporation on SCE induction by radiation, the comparative frequency of SCE and chromosome breaks induced by gamma radiation, the ability of ionizing radiation to interfere with normal cellular proliferation, and the relationship between proliferative inhibition and SCE and chromosome break frequency. A direct relationship between the number of SCEs and gamma radiation dose was observed, an effect which was dependent on BrdUrd incorporation. The frequency of SCE was 80 times higher than that of chromosome aberrations in cells with BrdUrd-substituted DNA, and there was no difference between the frequency of SCE in cells with or without chromosome breaks. The mitotic delay increased with the time between irradiation and harvesting. There was no relationship between the extent of mitotic delay and the number of SCEs.