Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in B6C3F1/Tk+/– mice treated neonatally with zidovudine and lamivudine

Linda S. Von Tungeln1, L. Patrice Hamilton1, Vasily N. Dobrovolsky2, Michelle E. Bishop2, Joseph G. Shaddock2, Robert H. Heflich2 and Frederick A. Beland1,3

1 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079 and
2 Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mother-to-child transmission of the human immunodeficiency virus is substantially reduced by prenatal and postnatal treatment with anti-retroviral nucleoside analogues; however, the long-term consequences of these drug interventions are not known. The nucleoside analogue zidovudine (3'-azido-2',3'-dideoxythymidine; AZT) is carcinogenic in mice when administered transplacentally or neonatally, and this may be due to a genotoxic mechanism. Since single-drug treatment with AZT is being superseded by multidrug combinations, we have investigated the induction of mutations and micronuclei in mice treated neonatally with AZT, lamivudine (3'-thia-2',3'-dideoxycytidine; 3TC), or a combination of the two drugs. B6C3F1/Tk+/– mice were treated daily from days 1–8 of age with 200 mg AZT/kg/day, 200 mg 3TC/kg/day, or a mixture of 200 mg AZT + 200 mg 3TC/kg/day (AZT/3TC). One and 2 days after the last dose, bone marrow was collected to assess the induction of micronuclei in polychromatic erythrocytes; 3 weeks following treatment, the induction of mutants was determined in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and thymidine kinase (Tk) genes of spleen lymphocytes. AZT and AZT/3TC, but not 3TC, caused a significant increase in micronuclei, with the response being greatest one day after the last dose. None of the drugs induced mutations in the Hprt gene, while AZT and AZT/3TC, but not 3TC, caused a significant increase in the Tk mutant frequency. The increase in Tk mutants by AZT and AZT/3TC was associated with loss of the wild-type (Tk+) allele (loss of heterozygosity). These data suggest that AZT, but not 3TC, is genotoxic in neonatal mice, and that 3TC does not alter significantly the responses observed with AZT alone.

Abbreviations: AIDS, acquired immunodeficiency syndrome; ANOVA, analysis of variance; APrC, adenine phosphoribosyltransferase (gene); AZT, 3'-azido-2',3'-dideoxythymidine, zidovudine; BrdUR, 5-bromodeoxyuridine-resistant; ddI, 2',3'-dideoxyinosine, didanosine; DMSO, dimethylsulfoxide; ENU, N-ethyl-N-nitrosourea; HIV, human immunodeficiency virus type 1; Hprt, hypoxanthine-guanine phosphoribosyltransferase (gene); LOH, loss of heterozygosity; PCE, polychromatic erythrocytes; PCR, polymerase chain reaction; PND, postnatal day; SEM, standard error of the mean; Tk, thymidine kinase (gene); 3TC, 3'-thia-2',3'-dideoxycytidine, lamivudine; 6-TGR, 6-thioguanine-resistant


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Women of childbearing age constitute nearly half of the 35 million adults worldwide who have human immunodeficiency virus type 1 (HIV) infection and acquired immunodeficiency syndrome (AIDS; ref. 1). In the absence of medical intervention, ~25% of children born to HIV-positive women will become infected (2,3). Given the magnitude of the worldwide epidemic, this has resulted in ~600 000 babies being born each year with the virus (1).

Mother-to-child HIV transmission can be significantly reduced by treatment with zidovudine (3'-azido-2',3'- dideoxythymidine; AZT), a nucleoside analogue that inhibits viral reverse transcriptase. When AZT is given after 14 weeks of pregnancy, intravenously during labor, and to newborns for the first 6 weeks of life, the frequency of HIV transmission drops from 25% to 8% (4,5). Based upon these results, this three-part AZT chemoprophylaxis regimen is recommended for all pregnant women infected with HIV (6).

A potential concern regarding the use of AZT is its reported tumorigenicity in mice. When given orally to CD-1 mice during the last third of gestation, AZT induced increases in lung, liver, ovary, mammary gland, and seminal vesicle tumors in the offspring (7,8). Likewise, when administered to neonatal mice on postnatal day (PND) 1 through 8, there was a significant dose-related trend in the incidences of lung and liver tumors in the female mice (8). In addition to being carcinogenic in mice, AZT is incorporated into the DNA of experimental animals (7,9–11).

While AZT treatment remains one of the few proven regimens for preventing mother-to-child transmission of HIV, AZT by itself has been shown to be less effective than combination therapies for treating HIV in adults (12,13). Consequently, most HIV-infected women are receiving two nucleoside reverse transcriptase inhibitors [e.g., AZT and lamivudine (3'-thia-2',3'-dideoxycytidine; 3TC)] and a protease inhibitor (14) when becoming pregnant. These women are then presented with the option of remaining on the same drug combination or discontinuing treatment until week 14 of pregnancy at which time they are typically given either AZT alone (6) or in combination with 3TC (6,15). Upon delivery the infants are also administered these drugs for periods of up to 6 weeks (6,15). The long-term risks or benefits of multi-drug treatment for either the mothers or their children are not known.

The incorporation of AZT into the DNA of target organs suggests that the tumors induced in mice are due to a genotoxic mechanism. Since single-drug treatment with AZT is being superseded by multidrug combinations, we have investigated the induction of mutations and micronuclei in mice treated neonatally with AZT, 3TC, or a combination of the two drugs.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Chemicals
AZT and 3TC were obtained from Glaxo Wellcome Oncology, Research Triangle Park, NC. N-Ethyl-N-nitrosourea (ENU) was purchased from Sigma Chemical Co., St Louis, MO.

Animals
Transgenic mice heterozygous for the thymidine kinase (Tk) gene were created using an embryonic stem cell line in which one allele of the endogenous Tk gene was inactivated by targeted homologous recombination (16). The Tk+/– genotype has been gradually transferred to a C57Bl/6N background by breeding Tk+/– males to C57Bl/6N females and genotyping the offspring by a three-primer allele-specific polymerase chain reaction (PCR) as described in Dobrovolsky et al. (17). Female C57Bl/6N/Tk+/– mice were mated with male C3H/HeNMTV mice to generate the B6C3F1/Tk+/– mice used in this study.

Treatment of B6C3F1/Tk+/– neonatal mice
Male and female B6C3F1/Tk+/– mice were treated daily on PND 1–8 with total doses of 4.0 mg AZT, 4.0 mg 3TC, or a mixture of 4.0 mg AZT and 4.0 mg 3TC. The compounds were dissolved in a mixture of dimethylsulfoxide (DMSO) and water (50/50; v/v), and administered in a 5 µl volume by intraperitoneal injection at a rate of 200 mg/kg/day. A single injection of 167 µg ENU in 5 µl aqueous DMSO was administered at PND 8 to serve as a positive control. Solvent control mice received daily injections of 5 µl aqueous DMSO. Five separate assays were conducted. Each assay included at least one nucleoside analogue group, a solvent control group, and a group treated with ENU.

Analysis of micronuclei
One and two days after the last dose (PND 9–10), bone marrow was collected, applied to microscope slides, fixed, and stained with acridine orange as described by Tinwell and Ashby (18). Micronuclei were scored, without knowledge of the treatment, in 1000 polychromatic erythrocytes (PCE) from each mouse.

In vivo mutagenesis
Three weeks after the last treatment (PND 28), the mutant frequency was determined in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and Tk genes of spleen lymphocytes. Assays for the frequency of Hprt mutants, as indicated by resistance to 6-thioguanine, and Tk mutants, as determined by resistance to 5-bromodeoxyuridine, were conducted as described by Meng et al. (19) and Dobrovolsky et al. (17), respectively, with the exceptions that the concentration of concanavalin A was increased to 6 µg/ml for priming the lymphocytes and an automated scoring technique (20) was used for scoring clones in both the Hprt and Tk assays. In instances where no Tk mutants were detected, the mice were re-genotyped using a new tissue sample to insure that the initial Tk+/– designation was correct. Loss of heterozygosity (LOH) analysis of 5-bromodeoxyuridine-resistant (BrdUR) lymphocyte clones was conducted by allele-specific PCR as outlined in ref. 17, using the primers designated TK14, TK16, and NEO4.

Statistical analyses
Statistical analyses of mutant frequency and micronuclei data were conducted by two-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. Data are reported as the mean ± standard error of the mean (SEM). When necessary, the data were transformed before the analyses to maintain an equal variance, normal distribution, or both. If a suitable transformation could not be found, the analyses were conducted using Kruskal–Wallis one-way ANOVA on ranks followed by Dunn's test. Analyses of survival, LOH, and intragenic mutation frequencies were conducted by {chi}2 tests. Bonferonni-type adjustments (21) were applied to correct for multiple comparisons.


    Results
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 Materials and methods
 Results
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 References
 
Hprt mutant frequency
Male and female B6C3F1/Tk+/– mice were treated daily on PND 1–8 with AZT, 3TC, or a mixture of AZT and 3TC. Thirty-nine percent of the mice treated with the solvent DMSO died during the treatment period, with the majority of deaths occurring during the first 3 days of treatment. The percent mortality in mice treated with AZT was similar (43%); 3TC mice showed lower mortality (22%; P = 0.003), while mice given both AZT and 3TC had a higher death rate (56%; P < 0.001).

Three weeks after the last treatment, the Hprt mutant frequency was assessed in spleen T-lymphocytes. The cloning efficiency of the T-lymphocytes was not affected by any of the treatments (Table IGo). Likewise, compared with mice administered the solvent alone, the mutant frequency, as measured by the number of 6-thioguanine-resistant (6-TGR) lymphocytes, was not increased upon treatment with AZT, 3TC, or the mixture of AZT and 3TC (Table IGo and Figure 1AGo). A substantial increase in the mutant frequency was observed with ENU, which served as a positive control (Table IGo and Figure 1AGo).


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Table I Cloning efficiency, 6-TGR mutant frequency, and BrdUR mutant frequency in B6C3F1/Tk+/– mice treated with AZT, 3TC, or a mixture of AZT and 3TCa
 


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Fig. 1. Hprt (A) and Tk (B) mutant frequency in male and female B6C3F1/Tk+/– mice treated daily on PND 1–8 with the solvent (aqueous DMSO), AZT, 3TC, or a mixture of AZT and 3TC (AZT/3TC), or on PND 8 with ENU. The mutant frequency was assessed 3 weeks after the last dose. The data are expressed as the mean ± SEM. *Indicates a significant difference (P < 0.05) from the control group.

 
Tk mutant frequency
The Tk mutant frequency was also assessed in the spleen T-lymphocytes three weeks after the last treatment. A two-way ANOVA indicated a significant (P < 0.001) treatment effect and a marginal (P = 0.077) sex effect. Compared with the solvent controls, dosing with AZT and a mixture of AZT and 3TC significantly increased the Tk mutant frequency, as indicated by the frequency of BrdUR lymphocytes (Table IGo and Figure 1BGo). Treatment with 3TC alone did not increase the mutant frequency. The positive control ENU gave a significant induction of Tk mutants. In a separate experiment, Tk and Hprt mutant frequencies were assessed 5 weeks after treatment with 3TC. Compared with the solvent controls, 3TC did not increase the mutant frequencies (Table IGo). As with the 3-week time point, ENU gave a substantial induction of Tk and Hprt mutants.

LOH analysis of BrdUR lymphocyte clones
The Tk gene of BrdUR clones was analyzed for LOH using three-primer allele-specific PCR. Representative examples are shown in Figure 2Go and the data are summarized in Table IIGo. The PCR results are divided into three classes. In instances where the intensity of the wild-type (Tk+) allele was greatly diminished compared with the disrupted (Tk-) allele, the clone was classified as having LOH. When the alleles were of similar intensity, the clone was considered to contain an intragenic mutation, for example, a point mutation or frameshift. When a designation could not be made, which occurred ~5% of the time, the clone was classified as `other'. Compared with the clones obtained from mice treated with the solvent alone, BrdUR clones from mice administered AZT or the mixture of AZT and 3TC had a significantly greater percentage of LOH and a corresponding decrease in intragenic mutation when assessed 3 weeks after the last treatment. The distribution of LOH and intragenic mutation in BrdUR clones from mice treated with 3TC alone did not differ from the respective control when assessed either 3 or 5 weeks after the last treatment.



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Fig. 2. Allele-specific PCR analysis of the Tk gene in BrdUR lymphocyte clones derived from B6C3F1/Tk+/– mice. The structure of the wild-type (Tk+) and disrupted (Tk-) alleles and the location of the primers used for the allele-specific PCR are shown. Clones in which the low-molecular-weight band corresponding to the Tk- allele was amplified preferentially were designated as LOH; clones in which the Tk- and Tk+ bands were amplified to a similar extent were classified as intragenic mutation. The following controls were included: clones sensitive to 5-bromodeoxyuridine (BrdUS), in which no amplification occurred, and clones from the cloning efficiency plates (CE+) and 6-TGR clones, in which both bands were amplified. M = 100 base pair ladder.

 

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Table II Loss of heterozygosity (LOH) analysis of BrdUR clones in B6C3F1/Tk+/– mice treated with AZT, 3TC, or a mixture of AZT and 3TCa
 
Analysis of micronucleated PCE
The induction of micronuclei in PCE was assessed on PND 9, which was 1 day after the last dose (Figure 3AGo). The response did not differ between sexes. Compared with the solvent controls, the administration of AZT or a mixture of AZT and 3TC significantly increased the frequency of micronucleated PCE. Treatment with 3TC alone did not increase the frequency of micronuclei. Likewise, the response observed with the mixture of AZT and 3TC did not differ significantly from that induced by AZT alone.



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Fig. 3. Frequency of PCE with micronuclei in male and female B6C3F1/Tk+/– mice treated daily on PND 1–8 with AZT, 3TC, or a mixture of AZT and 3TC (AZT/3TC). Assays were conducted (A) 1 (PND 9) and (B) 2 (PND 10) days after the last dose. The data are expressed as the mean ± SEM of 3–5 mice per sex per group. *Indicates a significant difference (P < 0.05) from the control group.

 
When measured on PND 10, the frequency of micronucleated PCE in mice treated with AZT or the mixture of AZT and 3TC decreased by >80% (P < 0.05) compared with that observed at PND 9 (Figure 3BGo). Nonetheless, the same trends were still evident: the frequency of micronuclei detected with AZT and the mixture of AZT and 3TC was significantly greater than that found with the controls and 3TC; the mixture of AZT and 3TC did not differ from AZT alone; and there was no difference between sexes. In addition, the response detected in the control and 3TC-treated mice did not differ between the 2 days.


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Sussman et al. (22) have reported that treatment of TK6 human B lymphoblastoid cells with AZT resulted in a 1.8-fold increase in the HPRT mutant frequency. This increase was associated with an increase in total gene deletions (rather than point mutations or partial gene deletions) and was correlated with the incorporation of AZT into genomic DNA. In a subsequent study, the mutant frequency was determined in the TK gene of TK6 cells exposed to AZT (23). A dose-related increase in the mutant frequency was observed, with the response being 2.2-fold greater in the TK gene compared with the HPRT gene. Molecular analyses indicated that 84% of the TK mutants were due to LOH, a property consistent with the DNA chain terminating characteristics of AZT. As before, the mutant frequency was correlated with the incorporation of AZT into genomic DNA. Similar experiments have also been conducted in AZH1 human B lymphoblastoid cells (24), using mutation induction in the adenine phosphoribosyltransferase (APRT) gene as the endpoint. In that study, AZT again gave a dose-related increase in the mutant frequency, and this was associated with LOH and the incorporation of AZT into genomic DNA. Mutant induction was not assessed in vivo; nonetheless, the administration of AZT in adult mice has been correlated with an increase in micronuclei and chromosomal aberrations (reviewed in ref. 25), genotoxic damage consistent with the DNA chain terminating properties of this nucleoside analogue.

The experiments reported here complement and extend these previous studies; thus, daily treatment with AZT on PND 1–8, a regimen shown to induce liver and lung tumors in mice (8), induced a three-fold increase in the Tk mutant frequency of spleen lymphocytes (Figure 1BGo). The Tk mutants from AZT-treated mice had a high frequency of LOH (Table IIGo), suggesting that the AZT was incorporated into the DNA and resulted in DNA chain termination with concomitant large-scale genetic alterations. The mutant frequency was not increased at the Hprt gene (Figure 1AGo), which because of its location on the X-chromosome is more suitable for detecting relatively small genetic changes, such as base substitutions and frameshifts. These results reinforce the concept that mutations induced by AZT are due to its ability to cause DNA chain termination. This interpretation is supported by the observation that AZT caused a substantial increase in bone marrow micronuclei (Figure 3Go), as has been reported previously in adult B6C3F1 mice (26).

AZT is metabolized to a mutagen through sequential phosphorylation to AZT 5'-triphosphate, which when incorporated into DNA will result in chain termination. The initial step in this activation involves conversion to AZT 5'-phosphate, a transformation catalyzed by thymidine kinase (27,28). Since Tk is one of the targets used in the mutation assays, it is conceivable that the increase in the Tk mutant frequency from treatment with AZT could be due to selection of pre-existing Tk-/- mutants. The available data suggest, however, that this is not the case and that the increase in the Tk mutant frequency is due to the de novo induction of mutants. The strongest evidence for this conclusion comes from the fact that the mutants detected from treatment with AZT have nearly twice the frequency of LOH as the spontaneous mutation spectra (Table IIGo). The selection of pre-existing Tk-/- mutants should amplify the spontaneous mutation spectra, while the induction of mutants should produce an agent-specific spectra, as was the case. Pre-existing Tk-/- mutants might be expected to have higher plating efficiencies than Tk+/- cells because they would be spared the toxic effects of AZT; however, again, this was not the case because the cloning efficiencies did not differ amongst the treatments (Table IGo). In addition, the survival of neonatal mice treated with AZT did not differ from those treated with the solvent alone.

There was a marginal sex-related difference in the AZT-induced Tk mutant frequency, with males being more sensitive. In preweanling male mice administered ENU, maximum mutation induction in the Hprt gene of spleen T-lymphocytes occurs ~3 weeks following treatment (29), the time point used in the present experiments. The observed mutant frequency is related to the age of the animal and has been attributed to an age-dependent trafficking of mutant cells from bone marrow and thymus to the spleen (29). It is presently unclear if there are sex-related differences in the trafficking of the mutants in neonatal mice. A sex difference was not apparent in the induction of micronuclei.

No increase in the Hprt (Figure 1AGo) or Tk (Figure 1BGo) mutant frequency was observed upon treatment with 3TC. Likewise the distribution of Tk mutations (i.e. LOH, intragenic, and other; Table IIGo) was similar in control mice and those treated with 3TC. The difference in response between AZT and 3TC may be a function of their molecular structures. AZT, being a thymidine analogue, is incorporated into DNA (7,9–11,22,23,25). 3TC is the L-enantiomer of a dideoxynucleoside analogue of cytidine. Although DNA polymerases catalyze the incorporation of both AZT and 3TC into DNA (30–32), 3TC, but not AZT, is rapidly excised by the 3'-5' exonuclease activity of the enzyme (31,32). This facile removal could explain the lack of mutation induction in the current experiment and also previous observations that 3TC is not mutagenic in microbial mutagenesis assays and is only weakly genotoxic in cultured human lymphocytes and in the mouse lymphoma assay (33). These results differ from those of Olivero et al. (34), who recently reported the incorporation of 3TC into the DNA of the fetuses of monkeys treated transplacentally with AZT and 3TC. In their study, however, there was considerable variation between animals (>100-fold in some instances), which suggests that additional experiments will be required before definitive conclusions can be drawn concerning the incorporation of 3TC into DNA in vivo.

Meng et al. (35) reported that the mutagenicity of AZT in TK6 B lymphoblastoid cells was potentiated by the nucleoside analogue didanosine (2',3'-dideoxyinosine, ddI). The increased mutant frequency was observed in both the HPRT and TK genes, and was attributed to an increase in incorporation of AZT into the lymphoblastoid cell DNA. In contrast to the in vitro results in human cells of Meng et al. (35), the Tk mutant frequency in B6C3F1/Tk+/– mice administered the mixture of AZT and 3TC was not greater than that observed with AZT alone (Figure 1BGo). In addition, 3TC did not augment the induction of micronuclei over that observed by AZT alone (Figure 3Go). We conclude, therefore, that AZT, but not 3TC, induces mutations and micronuclei in neonatal mice, and that 3TC does not attenuate or potentiate the response observed with AZT.


    Notes
 
3 To whom correspondence should be addressed at: HFT-110, National Center for Toxicological Research, Jefferson, AR 72079, USA Email: fbeland{at}nctr.fda.gov Back


    Acknowledgments
 
We thank Carey Nobles, Sherry Smith, and Crystal Thomas for assisting with the animal care and treatment, Goncialo Gamboa da Costa and Roberta Mittelstaedt for helping with some of the mutagenesis assays, and Cindy Hartwick for helping prepare the manuscript.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 21, 2001; revised May 13, 2002; accepted June 3, 2002.