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Genotoxicity kinetics in murine normoblasts as an approach for the in vivo action of difluorodeoxycytidine

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Abstract

Purpose

This study analyzed the kinetics of in vivo micronucleus induction in normoblasts by determining the kinetics of difluorodeoxycytidine (dFdC)-induced micronucleated polychromatic erythrocytes (MN-PCEs) in the peripheral blood of mice. The kinetic indexes of MN-PCE induction of dFdC were correlated with the previously reported mechanisms DNA damage induction by this compound. In general, this study aimed to establish an in vivo approach for discerning the processes underlying micronucleus induction by antineoplastic agents or mutagens in general.

Methods

The frequencies of PCEs and MN-PCEs in the peripheral blood of mice were determined prior to treatment and after treatment using dFdC at doses of 95, 190, or 380 µmol/kg at 8 h intervals throughout a 72 h post-treatment.

Results

The area beneath the curve (ABC) for MN-PCE induction as a function of time, which is an index of the total effect, indicated that the dose response was directly proportional and that the effect of dFdC on micronucleus induction was reduced compared with that of aneuploidogens and monofunctional and bifunctional alkylating agents but increased compared with that of promutagens, which is consistent with our previous results. The ABC showed a single peak with a small broadness index, which indicates that dFdC has a single mechanism or concomitant mechanisms for inducing DNA breaks. The time of the relative maximal induction (T rmi) indicated that dFdC requires more time to achieve MN-PCE induction compared with aneugens and monofunctional and bifunctional alkylating agents, although it requires a similar time to achieve MN-PCE induction as azacytidine, which is consistent with evidence showing that both agents must be incorporated into DNA for their action to be realized. The timing of maximal cytotoxicity observed with the lowest dFdC dose was correlated with the timing of the main genotoxic effect. However, early and late cytotoxic effects were detected, and these effects were independent of the genotoxic response.

Conclusions

A correlation analysis indicated that dFdC appears to induce MN-PCEs through only one mechanism or mechanisms that occur concomitantly, which could be explained by the previously reported concurrent inhibitory effects of dFdC on DNA polymerase alpha, polymerase epsilon, and/or topoisomerase. The timing of maximal cytotoxicity was correlated with the timing of maximal genotoxicity; however, an early cytotoxic effect that appeared to occur prior to the incorporation of dFdC into DNA was likely related to a previously reported inhibitory effect of dFdC on thymidylate synthase and/or ribonucleotide reductase.

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Acknowledgements

We would like to thank Angel Reyes and Perfecto Aguilar for providing excellent technical assistance.

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Authors and Affiliations

  1. Instituto Nacional de Investigaciones Nucleares, Apartado Postal 18-1027, Mexico City, Mexico

    Pedro Morales-Ramírez, Teresita Vallarino-Kelly & Virginia Cruz-Vallejo

Authors
  1. Pedro Morales-Ramírez
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  2. Teresita Vallarino-Kelly
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  3. Virginia Cruz-Vallejo
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Corresponding author

Correspondence to Pedro Morales-Ramírez.

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Animal welfare

The animals were treated and housed in accordance with the Guide for the Care and Use of Laboratory Animals, Commission on Life Sciences, Institute of Laboratory Animal Research, National Research Council (1996). The protocol was reviewed and approved by the Internal Committee of Care and Use of Laboratory Animals (CICUAL) that oversees the ethics of research involving the use and welfare of animals.

Funding

This work was supported by Project CB-507 from the Instituto Nacional de Investigaciones Nucleares (México) and Project 240116 from the Consejo Nacional de Ciencia y Tecnología (CONACYT) of México.

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Morales-Ramírez, P., Vallarino-Kelly, T. & Cruz-Vallejo, V. Genotoxicity kinetics in murine normoblasts as an approach for the in vivo action of difluorodeoxycytidine. Cancer Chemother Pharmacol 79, 843–853 (2017). https://doi.org/10.1007/s00280-017-3290-0

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