Clofarabine

IN VIVO KINETICS OF THE GENOTOXIC AND CYTOTOXIC ACTIVITIES OF CLADRIBINE AND CLOFARABINE

In vivo geno- and cytotoxic kinetics of cladribine and clofarabine

Key words: Micronucleus assay, normoblasts, genotoxicokinetics, cytotoxicokinetics, cytometry

Abstract

The aim of the present in vivo study was to determine the kinetics of the genotoxic and cytotoxic activities of cladribine and clofarabine in mouse normoblasts using flow cytometry. Mice in groups of five were treated with cladribine or clofarabine. Blood samples were obtained from the mouse tails before treatment and every 8 h posttreatment for 72 h. These samples were cytometrically scored for micronucleated reticulocytes (MN- RETs), and the percentage of reticulocytes was determined. The results showed that clofarabine and cladribine have early cytotoxic effects that are related to the genotoxic effects reported in previous studies; , the drugs have both complex long-lasting genotoxic and cytotoxic kinetic activity, with similar profiles that suggest a relationship between the genotoxic and cytotoxic parameters. The initial genotoxkinetics timing of clofarabine is equivalent to those of difluorodeoxycytidine, likely because both agents inhibit DNA polymerase. Clofarabine shows a higher genotoxic and cytotoxic efficiency than cladribine, in agreement with previous results.

Introduction

Not much information regarding the long-term genotoxic and cytotoxic actions in mammals after in vivo exposure to mutagens, specifically antineoplastic agents, is available. However, the time and duration of these genotoxic and cytotoxic effects, as well as their consequences on normal cells, is of interest for various conditions in which the entire organism is exposed [Hentosh and Peffley 2010]. Using microscopy, we have systematically studied the genotoxic and cytotoxic action of different types of agents in mouse normoblasts in vivo, monitoring the induction of micronuclei in young erythrocytes and the reduction in the frequency of these kinds of erythrocytes [Morales-Ramírez et al. 2014]. We observed that aneugens cause earlier induction than do mono and bifunctional alkylating agents, particularly those that require metabolic activation. Alkylating agents usually have single peak curves, with few exceptions, such as busulfan [Morales-Ramírez et al. 2004a]. We also studied two antimetabolites derived from cytidine, difluorodeoxycytidine, which has regular bell-shaped kinetics [Morales-Ramírez et al. 2017], and azacytidine, which has irregularly shaped kinetics and produces micronuclei for a very long time [Morales-Ramírez et al. 2008]. This is most likely correlated with the complex action of this agent leading to the formation of a mega-adduct complex by the covalent bonds between DNA containing azacytidine and DNA methyltransferase [Santi et al. 1984], which can subsequently cause breaks during repair [Hegde et al. 1996], the induction of demethylation [Jones and Taylor, 1980] and chromosomal fragility at GC-rich clusters [Satoh et al. 2004].

Cladribine and clofarabine are halogenated derivatives of adenine nucleotides used in the treatment of hematological malignancies. Clofarabine is a second-generation antineoplastic that has greater chemical and metabolic stability than exhibited by cladribine [Lotfi et al. 1999]. In models in vitro, both agents have shown a variety of effects, such as early cytotoxicity [Çelik et al. 2018], nucleotide reductase inhibition [Parker et al. 1991], DNA polymerase inhibition [Parker et al. 1991], hypomethylation [Zhang et al. 2009], and apoptosis induction [Genini et al. 2000]. In the present study, the complexity and duration of the kinetics of genotoxic and cytotoxic activity of cladribine and clofarabine were determined in mouse normoblasts in vivo as measured by flow cytometry.

Materials and Methods

Chemicals

Cladribine (2-chlorodeoxyadenosine, cat. Ab142414, purity>98%) and clofarabine (2-chloro-2’- arabino fluoro-2’-deoxyadenosine, cat. Ab142732, purity >98%) were purchased from Abcam Chemicals (Cambridge, MA, USA), and 1000 U/ml sodium heparin was obtained from Inhepar Laboratorios (Pisa, México). The anti-CD71-FITC antibody was obtained from BioLegend (cat. 334104, San Diego, CA, USA). Propidium iodide (cat. 02195458) and 1 mg/ml ribonuclease A (cat. 02101076) were obtained from MP Biomedicals, Inc. (Illkirch-Graffenstaden, France). BBS (bicarbonate-buffered solution) was obtained from Sigma (cat. S6297 and cat. S3014, respectively, St. Louis, MO, USA).

Animals

Male ICR mice weighing 30 g each were purchased from Envigo (Harlan, México), maintained at 22-24°C with 12 h dark-light periods; fed Purina Small Rodent Chow; and provided water ad libitum. The animals used in this study were treated and housed in accordance with the Guide for the Care and Use of Laboratory Animals by the 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), which oversees the ethics of research involving the use and welfare of animals.

Treatments

The mice in groups with five animals in each were intraperitoneally injected with 190 µmoles/kg cladribine or 100 µmoles/kg clofarabine diluted in 0.2 ml of water.

Samples

Blood samples were obtained from the tail of each mouse before (control) and every 8 h after treatment with cladribine or clofarabine for 72 h. The sampling times were established according to our previous studies [Morales-Ramírez et al. 2017b].

Sample fixing, labeling and analysis

The RET and MN-RET scoring of the blood samples was conducted using a cytometry method [Dertinger et al. 1996] according to the instruction for the MicroFlowPLUS kit (mouse peripheral blood from Litron Laboratories, Rochester, NY, MicroFlow-PLUS-Mouse-Blood.aspx). The blood samples were fixed, labeled and analyzed according to previously reported methods [Cervantes-Rios et al. 2012] and modified for the low volume blood samples obtained from the mice. Samples of 30 µl of blood were obtained from the tail of each animal and mixed with 30 µl of sodium heparin. These samples were then diluted 1:2 with sodium bicarbonate-buffered solution (BBS) at pH 7.5 (0.9 g of NaCl and 0.0444 g of NaHCO3 diluted in 100 ml of distilled water), of which a 100 µl sample was fixed in a cryotube with 2 ml of methanol at -75°C by rapid vortex mixing. To improve the efficiency and reproducibility of the fixation procedure, a 1.5 ml Eppendorf tube was cut at the extreme end and used as a guide for the micropipette tip used for transferring the sample into the 4.5 ml fixation tube. This precision allowed the sample to be deposited in the center of the fixation tube within a short distance of the vortex produced by an electric mixer in -75°C methanol.

The use of this dispositive method enabled the correct fixation of 100% of the samples and an analysis of 90% of the cells with minimal number of cell aggregates [Cruz-Vallejo et al. 2019]. The fixed samples were immediately stored at -75°C until labeling and staining for analysis. One milliliter of the fixed sample was mixed with 7 ml of cold BBS and centrifuged at 600 × g at 4°C for 5 min. The pellet was resuspended in 25 µl of BBS and mixed with 80 µl of ribonuclease A in a polystyrene tube. Three types of tubes were prepared: i) autofluorescence reagent containing 25 µl of the sample; ii) control CD71-sample fluorescence reagent containing 25 µl of the sample and 5 µl of anti-CD71-FITC, which allowed us to distinguish the RETs; and iii) Samples containing 25 µl of sample with 5 µl of anti-CD71-FITC. The tubes were incubated at 4°C for 40 min and then at 22°C for 90 min. Finally, 500 µl of cold BBS was added to each tube, and the tubes were maintained in the dark at 4°C until analysis.

Two microliters of propidium iodide (820 µg/ml in cold BBS) was added to the (iii) sample tubes 15 min before the cells were scored with a cytometer. In a FACSCalibur instrument (Becton Dickinson) with Cell Quest software (Becton Dickinson, version 3.0.1), one million events were scored using an argon laser set at a 488 nm excitation wavelength at low speed. For all animals, at least 4000 RET were scored according to the Guidelines from the OCDE [OECD/OCDE. (2016) OECD Guideline for the testing of chemicals. Mammalian erythrocyte micronucleus test. TG 474]. The emissions from the anti-CD71-FITC and propidium iodide stains were detected in channels FL1 and FL2, respectively. The regions with RET and erythrocytes with and without treatment were discerned according to Cervantes et al. [2012]. The data were analyzed with WinMDI software version 2.9 (Joseph Trotter, [email protected]).

Genotoxicity

The genotoxicity caused by each treatment was established by the area beneath the curve (ABC) of the MN-RET frequency versus time, and the genotoxic efficiency was the ABC value per dose in units of µmoles/kg of bd wt.

Cytotoxicity

Cytotoxicity was determined as the reduction in the frequency of RETs versus time by calculating the reduction in the ABC of the RET number versus time. The total ABC was estimated assuming that the basal RET frequency was near constant over time.

Statistics

Samples obtained prior to treatment were used as the basal frequency for each animal and used in statistical comparisons with values for each post treatment time. We determined the data as parametric using the Shapiro-Wilks test. The differences at the posttreatment times were established by comparing the obtained value with the baseline value of the corresponding individual, and the significance was determined with a paired t-test, and p<0.05 indicated statistical significance. Results the MN-RET induction and cytotoxicity increase curves in terms of the reduction in the percentage of RETs as a function of time after mice were treated with 100 micromoles of clofarabine or 190 micromoles of clofarabine per kilogram of weight. Cladribine and clofarabine induced an increase in the MN-RETs from 16 to 64 h; although the increases were significant only for between 24 and 56 h. The area beneath the curve values for clofarabine indicated an almost two- fold enhanced induction with approximately one-half the dose of cladribine; that is, clofarabine was 4-fold more genotoxic than cladribine. Regarding cytotoxicity, cladribine showed increased cytotoxicity from 8 to 64 h, and the increase from 8 to 48 h was significant. In the case of clofarabine, an increase was observed from 8 to 64 h but was significant only between 8 and 56 h. In this case, the cytotoxicity of clofarabine was approximately 40% higher than that of cladribine The profiles of the genotoxicity and cytotoxicity curves were similar, with the exception of the 8 and 16 h time points, which be showed significantly greater cytotoxicity, while the genotoxicity is minimal comparisons of the profiles of the induction curves with cladribine and clofarabine with those obtained previously after exposure to azacytidine and difluorodeoxycytidine [Morales- Ramírez et al. 2008; 2017]. The profile of difluorodeoxycytidine has a simple, bell shape showing the MN-RET induction from 24 to 48, that is, for only approximately 24 h. Azacytidine has a complex profile, with induction shown beginning at 24 h and continuing through 72 h, that is, for more than 48 h. The profiles of cladribine and clofarabine are also complex and, as described, show MN-RET induction lasting 48 h. Discussion The induction of MN-RET in mouse normoblasts by chemical agents in vivo is a complex biological process that can be divided into at least three stages: i) pharmacokinetics, from the time of administration to when it reaches the target organ; ii) cellular processes, comprising the interaction of the agent with the cell, the production of breaks in the DNA and the formation of MN-normoblasts; iii) the expression stage, in which the DNA ruptures produces micronuclei in reticulocytes and includes the cell differentiation of the normoblasts into erythrocytes after enucleation and the appearance of RETs in peripheral blood. The pharmacokinetic stage in the case of nucleotide derivatives and other agents does not appear to have a significant effect on the time of MN-RET induction, with the exception of agents that require metabolic activation in the liver [Morales-Ramírez et al. 2014]. Similarly, the duration of the expression stage may be variable, but there is no evidence that it depends on the effect of the mutagens. Therefore, the differences in the temporal expression of micronuclei depend on the processes involved in the production of aneuploidy or breaks in the DNA. This timing can be explained in terms of the stage of the cell cycle at which the agent can act to cause DNA rupture or aneuploidy. The marrow continuously produces erythrocytes; therefore, there are normoblasts at different stages of the cell cycle. That is, the normoblasts that are currently undergoing mitosis are sensitive to aneugens and will produce isolated chromosomes and, subsequently, micronuclei in this mitosis [Morales-Ramírez et al. 2004b]. For example, if an agent must be incorporated into DNA to inhibit a polymerase [Morales-Ramírez et al. 2017], its effect will occur in the synthesis stage; however, the cell must resume and complete synthesis, enter into the G2 stage and undergo mitosis for MN-RET to be observed. Regarding the duration and shape of the kinetic curves, we have observed very simple bell-shaped kinetics with a duration of approximately 24 h [Morales-Ramírez et al. 2017] or complex irregularly shaped kinetic curves, such as that of azacytidine, with a long-term effect, more than 48 h [Morales- Ramírez et al. 2008]. The azacytidine profile is attributed to its complex action that subsequently generates a mega adduct complexes [Santi et al. 1984], and induces breaks during DNA repair [Hegde et al. 1996], demethylation [Jones and Taylor, 1980] and chromosomal fragility [Satoh et al. 2004]. The complex and prolonged kinetic responses obtained with cladribine and clofarabine can be attributed to the variety of actions that have been reported for these agents, and although the sequence of their actions can be inferred, the kinetics do not allow unequivocal certainty regarding the response and the mechanisms involved, i.e., the initial genotoxkinetics timing of clofarabine seem equivalent to those of dFdC, likely because both agents inhibit DNA polymerase [Parker et al. 1991; Huang et al. 1991]. Furthermore, some mechanisms may not have yet been reported, such as those associated with the early cytotoxicity caused by cladribine or clofarabine [Çelik et al. 2018], which has recently been reported, despite studies on these agents dating back three or four decades. The early cytotoxicity caused by these agents does not require the introduction of the agent into the cell, only their interaction with the CD99 membrane protein, which triggers cell death. Coincidentally, in the present study, early cytotoxicity was observed, almost 16 h before the genotoxic effect was manifested. Although there are no reports of the presence of CD99 in normoblasts, it has been found on erythrocytes [Latron et al. 1987]. The complex kinetics caused by azacytidine, cladribine and clofarabine show the need to deepen the knowledge of the phenomena triggered by these treatments in vivo through strategies that include molecular methods. The knowledge thereby generated can improve therapeutic protocols and be used to explain or predict the side effects of antineoplastic agents on healthy cells. Conclusions a. Clofarabine and cladribine have an early cytotoxic effect, not related to their genotoxic effect. b. The kinetics of genotoxic action caused by cladribine and clofarabine are complex and indicate a prolonged genotoxic effect for a period of 48 h. c. The kinetics of the cytotoxic action by cladribine and clofarabine are also complex and indicate cytotoxicity that lasts for 64 h. d. The profiles of the genotoxicity and cytotoxicity curves are similar, with the exception of an early and significant increase in cytotoxicity while genotoxicity remains minimal. e. Clofarabine has more genotoxic and cytotoxic effect than cladribine. f. The differences in the temporal expression of micronuclei depend on the processes involved during the induction of aneuploidy or DNA breaks. g. Concomitant genotoxicokinetics and cytotoxicokinetics analyses may be useful as a preclinical assays to establish the duration and complexity of the antineoplastic agent response in vivo. Author contributions All of the authors contributed to the conception and design of the study, the acquisition of data, the analysis and interpretation of data, the drafting of the article, the critical revisions of the article for important intellectual content, and the approval of the version to be published. Acknowledgments We wish to thank Miguel Angel García Torres and Faustino Antonio Anastacio Montes Ϯ for their excellent technical assistance. Sponsors This work was supported by project CB-240116-2014 from the Consejo Nacional de Ciencia y Tecnología (CONACYT, México) to Dr. Pedro Morales-Ramírez and scholarship 302735 for postgraduate studies to M.S. Jesús Quezada-Vidal. Conflict of interest statement All authors declare that there are no conflicts of interest. References Çelik H, Sciandra M, Flashner B, Gelmez E, Kayraklıoğlu N, Allegakoen DV, Petro JR, Conn EJ, Hour S, Han J, et al. 2018. Clofarabine inhibits Ewing sarcoma growth through a novel molecular mechanism involving direct binding to CD99. Oncogene 37:2181-2196. Cervantes-Rios E, Ortíz-Muníz R, Martínez-Hernández AL, Cabrera-Rojo L, Graniel-Guerrero J, Rodríguez-Cruz L. 2012. Malnutrition and infection influence the peripheral blood reticulocyte micronuclei frequency in children. Mutat Res 731:68-74. Commission on Life Sciences, Institute of Laboratory Animal Research, National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Cruz-Vallejo V, Ortíz-Muñíz R, Vallarino-Kelly T, Cervantes-Ríos E, Morales-Ramírez P. 2019. In vivo Characterization of the Radiosensitizing Effect of a very low dose of BrdU in murine cells exposed to low-dose radiation. Environ Mol Mutagen 60:534:545. Dertinger SD, Torous DK, Tometsko KR. 1996. Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer. Mutat Res. 371:283-292. Genini D, Adachi S, Chao Q, Rose DW, Carrera CJ, Cottam HB, Carson DA, Leoni LM. 2000. Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria. Blood 96:3537–3543. HegdeV, McFarlane RJ, Taylor EM, Price C. 1996. The genetics of the repair of 5-azacytidine- mediated DNA damage in the fission yeast. Mol Gen Genet 251:483-492. Hentosh P, Peffley DM. 2010. The cladribine conundrum: deciphering the drug's mechanism of action. Expert Opin Drug Metab Toxicol 6:75-81. Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. 1991. Action of 2',2'- difluorodeoxycytidine on DNA synthesis. Cancer Res 51:6110-6117. Jones PA, Taylor SM. 1980. Cellular differentiation, cytidine analogs and DNA methylation. Cell 20:85-93. Latron,F., Blanchard,D. and Cartron,J.P. 1987. Immunochemical characterization of the human blood cell membrane glycoprotein recognized by the monoclonal antibody 12E7. Biochem 247:757- 764. DOI: 10.1042/bj2470757 Lotfi K, Mansson E, Spasokoukotskaja T, Pettersson B, Liliemark J, Peterson C, Eriksson S, Albertioni F. 1999. Biochemical pharmacology and resistance to 2-chloro-2'-arabino-fluoro-2'- deoxyadenosine, a novel analogue of cladribine in human leukemic cells. Clin Cancer Res 5:2438- 44. Morales-Ramírez P, Vallarino-Kelly T, Cruz-Vallejo VL, López-Iturbe R, Alvaro-Delgadillo H. 2004a. In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics. Mutagenesis 19:207-213. Morales-Ramírez, P., Vallarino-Kelly, T., Cruz-Vallejo, V.L. 2004b. Kinetics of micronucleated polychromatic erythrocytes (MN-PCE) induction in vivo by aneuploidogens. Mutation Res. 565:79- 87. Morales-Ramírez P, Vallarino-Kelly T, Cruz-Vallejo VL. 2008. Mechanisms of DNA breaks induction in vivo by 5-azacytidine: paths of micronucleus induction by azaC. J Apply Toxicol 28:254-259. Morales-Ramírez P, Vallarino-Kelly T, Cruz-Vallejo VL. 2014. Kinetics of micronucleus induction and cytotoxicity caused by distinct antineoplastics and alkylating agents in vivo. Toxicol Lett 224:319–325. Morales-Ramírez P, Vallarino-Kelly T, Cruz-Vallejo VL. 2017. Genotoxicity kinetics in murine normoblasts as an approach for the in vivo action of difluorodeoxycytidine. Cancer Chemother Pharmacol 79:843–853. OECD/OCDE. (2016) OECD Guideline for the testing of chemicals. Mammalian erythrocyte micronucleus test. TG 474]. Parker WB, Shaddix SC, Chang CH, White EL, Rose LM, Brockman RW, Shortnacy AT, Montgomery JA, Secrist JA3rd, Bennett LLJr. 1991. Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta- D-arabinofuranosyl) adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5’-triphosphate. Cancer Res 51:2386–2394. Santi DV, Norment A, Garrett CE. 1984. Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci U S A. 81:6993-6997. Satoh T, Yamamoto K, Miura KF, Sofuni T. 2004. Region-specific Clofarabine chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells. Cytogenet Genome Res 104:289-294.

Zhang Y, Shahriar M, Zhang J, Ahmed SU, Lim SH. 2009. Clofarabine induces hypomethylation of DNA and expression of Cancer-Testis antigens. Leukemia Res 33:1678-1683.