Elevated frequencies of micronucleated erythrocytes in infants exposed to zidovudine in utero and postpartum to prevent mother-to-child transmission of HIV

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Environ Mol Mutagen. Author manuscript; available in PMC 2007 July 5.
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Published in final edited form as:
Environ Mol Mutagen. 2007 ; 48(3-4): 322–329.
Elevated frequencies of micronucleated erythrocytes in infants
exposed to zidovudine in utero and postpartum to prevent mother-
to-child transmission of HIV
Kristine L. Witt1, Coleen K. Cunningham2, Kristine B. Patterson3, Grace E. Kissling1,
Stephen D. Dertinger4, Elizabeth Livingston2, and Jack B. Bishop1,*
1 National Toxicology Program, National Institute of Environmental Health Sciences, RTP, NC
2 Duke University Medical Center, Durham, NC
3 University of North Carolina Department of Medicine, Chapel Hill, NC
4 Litron Laboratories, Rochester NY
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Abstract
Zidovudine-based antiretroviral therapies (ART) for treatment of HIV-infected pregnant women
have markedly reduced mother-to-child transmission of the human immunodeficiency virus (HIV-1)
from ~25% to <1%. However, zidovudine (ZDV; AZT), a nucleoside analogue, induces
chromosomal damage, gene mutations, and cancer in animals following direct or transplacental
exposures. To determine if chromosomal damage is induced by ZDV in infants exposed
transplacentally, we evaluated micronucleated reticulocyte frequencies (%MN-RET) in 16 HIV-
infected ART-treated mother-infant pairs. Thirteen women received prenatal ART containing ZDV;
3 received ART without ZDV. All infants received ZDV for 6 weeks postpartum. Venous blood was
obtained from women at delivery, and from infants at 1–3 days, 4–6 weeks, and 4–6 months of life;
cord blood was collected immediately after delivery. Ten cord blood samples (controls) were
obtained from infants of HIV-uninfected women who did not receive ART. %MN-RET was
measured using a single laser 3-color flow cytometric system. Ten-fold increases in %MN-RET were
seen in women and infants who received ZDV-containing ART prenatally; no increases were detected
in 3 women and infants who received prenatal ART without ZDV. Specifically, mean %MN-RET
in cord blood of ZDV-exposed infants was 1.67±0.34 compared with 0.16±0.06 in non-ZDV ART-
exposed infants (P=0.006) and 0.12±0.02 in control cord bloods (p<0.0001). %MN-RET in ZDV-
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exposed newborns decreased over the first 6 months of life to levels comparable to cord blood
controls. These results demonstrate that transplacental ZDV exposure is genotoxic in humans. Long-
term monitoring of HIV-uninfected ZDV-exposed infants is recommended to ensure their continued
health.
Keywords
AZT; antiretroviral; chromosome damage; transplacental exposure; nucleoside analogues;
mutagenicity
*correspondence: Jack B. Bishop, Ph.D.,National Institute of Environmental Health Sciences, Environmental Toxicology Program, PO
Box 12233, Mail Drop EC-01, Research Triangle Park, NC 27709, phone: 919-541-1876, fax: 919-361-4511, [email protected]
Conflict of interest statement
S Dertinger is employed by Litron Laboratories, which holds patents pertaining to the flow cytometric analysis of micronucleated
erythrocytes.
This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), and the NIEHS.

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INTRODUCTION
The nucleoside analogue zidovudine (ZDV) is widely used in combination antiretroviral
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therapy (ART) regimens and these regimens are highly effective in reducing mother-to-child
transmission of HIV from an estimated rate of 25% to less than 1% (Dorenbaum et al., 2002).
In the United States, to protect against vertical transmission, HIV-infected pregnant women
typically receive ART as standard care during pregnancy and their infants receive prophylactic
ZDV for 6 weeks postpartum (CDC, 2005). Thus, HIV-uninfected infants born to mothers with
HIV may be exposed to ZDV both transplacentally during gestation, and directly during the
postpartum treatment period. Although ZDV is tremendously effective in preventing vertical
transmission of HIV, ZDV has been shown to be carcinogenic in animals (Ayers et al.,
1996;National Toxicology Program, 1999,2006;Olivero et al., 1997;Diwan et al., 1999),
genotoxic in numerous in vitro and in vivo test systems (Phillips et al., 1991;Sussman et al.,
1999;Poirier et al., 2004;Chan et al., in press;von Tungeln et al., 2004;Meng et al., 2002), and
incorporated into DNA of primates and humans after transplacental or direct exposure (Poirier
et al., 2004,1999;Olivero et al., 2000,2002).
Of greatest concern to our group were results of recent experiments we conducted in mice that
revealed 10-fold increases in micronucleated reticulocytes (immature erythrocytes) in
peripheral blood of newborn pups exposed to a human equivalent dose of ZDV throughout
gestation and for the first 3 weeks of life; the treatment regimen in these mouse studies modeled
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that used in humans to prevent mother-to-child transmission of HIV (Bishop et al., 2004;Witt
et al., 2004). Micronuclei (MN) are well-characterized biomarkers of structural or numerical
chromosomal damage; they arise from acentric chromosome fragments or lagging whole
chromosome(s) that fail to incorporate into daughter nuclei after nuclear division (Heddle et
al., 1991), and they are commonly evaluated in lymphocytes and erythrocytes, due to the ease
of obtaining blood samples. Prompted by the observations of increased frequencies of
micronucleated reticulocytes (MN-RET) in mouse pups exposed to ZDV, we designed a pilot
study to determine if similar effects were detectable in human infants exposed to ZDV in utero
and postnatally. We used a recently developed single-laser three-color flow cytometric system
for enumerating MN-RET in human blood (Dertinger et al., 2004). The advent of this new
technology was important for our study design because, although the mouse peripheral blood
erythrocyte micronucleus test has been used routinely for decades to evaluate genotoxicity
(Heddle et al., 1991;OECD, 1997; MacGregor et al., 1990; Albertini et al., 2000;Witt et al.,
2000;Hayashi et al., 1994), there has not been a corresponding human assay because the human
spleen, unlike the mouse spleen, rapidly removes damaged erythrocytes from circulation. Thus,
until recently, experimental evidence of genotoxicity obtained through use of routine rodent
erythrocyte MN studies could not be confirmed in the same cell type in humans unless the
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human subject had undergone splenectomy (MacGregor et al., 1997). However, this new flow
cytometry-based test method enables the identification, selection, and evaluation of the
youngest fraction of reticulocytes (<48 hr old), representing approximately10% of all
circulating reticulocytes, before the effects of splenic selection have much impact on the
frequency of MN-RET. Identification of this fraction of reticulocytes is based on expression
of a specific cell surface marker, the transferrin (CD71) receptor, which is active for only a
few hours after the young reticulocyte enters the peripheral circulation. Thus, using this new
technology, we were able to minimize the action of the spleen on frequency of MN-RET and
directly compare a well-characterized biomarker of genotoxicity (i.e., MN) in the same cell
type (reticulocytes) in the animal model system and in humans.
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MATERIALS AND METHODS
Study Subjects
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All pregnant women in this study were patients at one of the two participating clinical sites,
Duke University Medical Center (DUMC) or University of North Carolina (UNC). Because
this was a pilot study, few restrictions were placed on subject eligibility. Cases were broadly
defined as pregnant women who were HIV-infected, receiving prenatal antiretroviral therapy
(ART); inclusion of ZDV in the ART was not specifically required. Detailed demographic
information was collected for each study subject, but there were no exclusions based on race,
ethnicity, age, HIV RNA, CD4 counts, or method of delivery (vaginal or caesarean). Controls
were defined as HIV-uninfected women with uncomplicated, full-term, singleton pregnancies.
The study protocol was approved by the Institutional Review Boards of the National Institute
of Environmental Health Sciences (NIEHS), DUMC, and UNC. All women provided written
informed consent.
Cases consisted of 16 HIV-infected pregnant women who received prenatal antiretroviral
therapy (ART) as prescribed by their HIV providers. Thirteen of these women received prenatal
ART that included ZDV as a component and 3 women received ART without ZDV (Tables I
and II). All 16 women received intravenous ZDV during labor and delivery, and all 16 infants
born to these women received prophylactic ZDV orally for 6 weeks postpartum, at standard
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doses. All infants were HIV-DNA PCR negative at birth and at 6 months of age.
Blood Sampling Schedule
All blood samples were obtained at routine, scheduled blood draws; no blood samples were
requested solely for this study. Therefore, maternal venous blood was collected at the time of
delivery and infant cord blood was obtained immediately after delivery. Venous blood samples
were obtained from infants at 1–3 days, 4–6 weeks, and 4–6 months of life, in conjunction with
routine, scheduled blood draws. Cord blood samples were also obtained from infants born to
10 HIV-uninfected women who were not receiving ART, and these served as controls.
MN-RET Measurements
MN-RET CD71+ frequencies (%MN-RET) were measured in heparinized venous blood samples
obtained from women and infants at the time points outlined above. Samples were processed
using flow cytometric micronucleus analysis kits (Prototype Human MicroFlow® kits, Litron
Laboratories, Rochester NY). Details of the collection, processing, and analysis of blood
samples using this method have been previously described (Dertinger et al., 2004). Briefly, a
60–120 μL sample of whole blood (venous or cord blood) was diluted with 350μL of sodium
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heparin solution. Aliquots of the heparinized blood samples were then forcefully injected into
tubes containing ultracold (−80°C) methanol at the clinical site. These fixed blood samples
were allowed to accumulate in a −80°C freezer and then were bulk-shipped overnight on dry
ice to Litron Laboratories for flow cytometric analysis. Approximately 20,000 young
reticulocytes (i.e., CD71-positive) were evaluated per blood sample for %MN-RET.
Statistical Analysis
Because %MN-RET values were often not normally distributed and because some of the
sample sizes in subgroups were small (i.e., the 3 non-ZDV-exposed women and infants), the
nonparametric one-sided Mann-Whitney test was used to compare two groups, and Kruskal-
Wallis analysis of variance was used to compare three or more groups. Spearman correlation
coefficients were used to relate % MN-RET values at each of the five sample times to
continuous variables such as maternal age, weight, CD4 counts, and HIV RNA.
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RESULTS
A detailed description of the study population is presented in Table I, while the complete %
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MN-RET data set for women and infants is shown in Table II. A few blood specimens were
not amenable to flow cytometric analysis and, thus, the numbers of samples analyzed in the
specific study groups discussed below do not always equal the number of subjects sampled.
Nevertheless, the positive genotoxic outcomes in ART-exposed patients described below
remained independent and significant when correlated to other variables. %MN-RET was
unrelated to maternal age, height, weight, body mass index, CD4 counts, or HIV RNA values
(p≥0.10); in addition, delivery method (vaginal or caesarean), baby’s sex, location of clinic,
duration of prenatal ART, birth weight, and gestational age were also unrelated to %MN-RET
(p≥0.10) (Table III). No correlation was seen between %MN-RET and any of the non-ZDV
medications (nucleosides as well as protease inhibitors) included in the prenatal ART regimens
represented among the 16 cases.
The mean MN-RET frequencies observed in the women and newborns (data from the cord and
day 1–3 infant bloods) who received prenatal ART that included ZDV were increased 10-fold
compared with the frequency in the control cord blood samples; the three mother-infant pairs
who received prenatal ART without ZDV did not show significant increases in MN-RET (Fig.
1). In cord blood (Fig. 1; Table II), the mean %MN-RET in newborns exposed to ZDV in utero
(N=12) was 1.67 ± 0.34 (p<0.0001, compared with controls). Mean %MN-RET in newborns
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not exposed to ZDV (N=3) in utero was 0.16 ± 0.06 (significantly lower than the ZDV-exposed
group, p=0.006; but not different from controls, p=0.17). The mean %MN-RET in cord blood
control samples (N=10) was 0.12 ± 0.02, which closely matches the normal values for %MN-
RET in a sample of 50 healthy male and female adults (0.12±0.009%) (Dertinger et al.).
Women who received ZDV as part of their prenatal ART therapy (N=11) had a mean %MN-
RET of 1.51 ± 0.62, which was significantly elevated (p=0.015) over the mean (0.12±0.02) for
women who did not receive prenatal ZDV (N=3). The mean %MN-RET for the women who
did not receive prenatal ZDV closely matches the mean %MN-RET reported for the sample
of 50 (25 males, 25 females) healthy adults referenced above (Dertinger et al.).
The markedly elevated %MN-RET in newborns exposed to ZDV in utero decreased during
the postpartum ZDV treatment period (Fig. 1). At the 4–6 week sampling time, the mean %
MN-RET in infants transplacentally exposed to ZDV had decreased to 0.45 ± 0.15, a value
that was still significantly elevated over the cord blood control frequency (p = 0.025). This
decline is consistent with the marked reduction in erythropoiesis that normally occurs in
newborns at birth and persists for approximately 6–8 weeks after birth (Palis and Segel,
1998). By age 6 months, %MN-RET (N=7) declined to levels comparable to control cord
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bloods (p=0.41) (Fig. 1; Table II); this observation is consistent with the completion of ZDV
prophylaxis well before the final postnatal blood sampling.
%MN-RET values obtained with cord blood and with infant venous blood sampled at 1–3 days
of age were essentially identical (Table II), suggesting that either sample type could be selected
for evaluation in future studies.
DISCUSSION
The nucleoside analogue ZDV is highly effective at reducing mother-to-child transmission of
HIV (Dorenbaum et al., 2002). As such, it is a critical component of antiretroviral treatment
regimens. However, data on adverse genetic effects induced by ZDV in a variety of test systems
are accumulating. Recently, increasing emphasis has been placed on examining the effects in
infants of transplacental exposures to ZDV due to the increasing number of children born to
HIV-infected women who receive ART during pregnancy. For example, experiments in mice
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have shown that transplacental exposure to ZDV results in an increased frequency of cancer
in exposed mice at maturity (NTP, 2006;Olivero et al., 1997), as well as increases in
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mitochondrial DNA mutations (Chan et al., in press) and elevated mutation frequencies in
lymphocytes (von Tungeln et al., 2002,2004,2007;Torres et al., 2007). Furthermore, the
incidences of K-ras and p53 mutations were found to be elevated in lung tumors arising in
adult mice transplacentally exposed to ZDV, compared with the incidences of these mutations
in spontaneous tumors arising in unexposed mice, suggesting that these mutations were the
result of chemical-specific events leading to increases in tumor formation later in life (Hong
et al., in press). In humans, ZDV has been shown to incorporate into DNA of cord blood cells
and maternal lymphocytes after prenatal and perinatal exposures (Olivero et al., 2000).
Our previous experiments in mice investigating the induction of chromosomal damage
following transplacental exposure to human equivalent doses of ZDV, alone or in combination
with other nucleosides, showed that ZDV induced extraordinary, 10-fold increases in
micronucleated erythrocytes (Bishop et al., 2004;Witt et al., 2004), implying a strong potential
for ZDV-induced genetic damage in all exposed dividing cell populations. To determine
whether similar effects could be detected in infants exposed to ZDV in utero as a consequence
of maternal ART, we designed and conducted the study reported here. We included an extended
monitoring period so that any effects that might be noted in infants at birth could be followed
during the 6-week postpartum treatment period and further out to 6 months of age, after all
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exposure to ZDV had ceased. The results in infants following transplacental exposure are in
accord with those obtained in the mouse model (10-fold increases in MN-RET were noted in
both infants and mouse pups) and suggest that, although we are monitoring a biomarker of
chromosomal damage in a surrogate population of terminally differentiated cells, other
dividing cell populations and stem cells that are exposed to ZDV may be at risk for acquiring
similar damage. The health consequences of this damage are not yet evident, and will depend
in large part on whether the ZDV-induced damage is eliminated by repair enzymes or cell
death, or converted to a stable, persisting alteration in a continuously propagating tissue. In
addition, since ZDV is a chain terminator (Zidovudine, IARC Monograph vol. 76, 2000) and
affects telomere length (Olivero et al., 1997), it is possible that consequences involving
accelerated aging or related effects may result from ZDV-induced chromosomal damage.
The flow cytometric system used to evaluate %MN-RET in this study has been validated in
mice (Torous et al., 2005) and is accepted by regulatory agencies as a method of measuring
genotoxicity. This study in HIV-infected women and their infants is one of the first to extend
this evaluation procedure to human blood samples. Results of similar studies in humans have
recently been reported (Stopper et al., 2005;Dertinger et al.;Harrod et al.) and all, including
the study reported here, have shown remarkable consistency in baseline MN-RET frequencies
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in adult and pediatric control populations (~0.12%) as well as a clear ability to detect and
quantify changes from baseline levels following exposures to known clastogens such as
chemotherapeutic agents or radiotherapy. In our study, we achieved remarkable consistency
between mean maternal and infant cord blood MN-RET frequencies, as well as highly similar
values in instances where duplicate samples were evaluated from a single blood draw (data not
shown), demonstrating that our measurements were reproducible.
Studies in human populations have shown that elevated lymphocyte chromosome aberration
frequencies, indicating exposure to a genotoxicant, are associated with an increased risk of
chronic diseases, particularly cancer, for that population (Albertini et al., 2000;Rossner et al,
2005;Bonassi et al., 2005). The frequency of micronucleated lymphocytes in healthy
individuals has also been directly correlated with an increased risk of future cancer (Bonassi
et al., in press). However, the degree of correlation between MN frequencies in lymphocytes
and in reticulocytes has only been partially characterized in a recent pilot study (Stopper et al.,
2005). Therefore, we cannot conclude with certainty that the elevated reticulocyte
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micronucleus frequencies observed in ZDV-exposed infants in our study will be associated
with a higher risk of future disease in these infants compared with healthy, non-exposed
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individuals. However, we are concerned about the long-term health implications for these
infants because the MN increases noted in this study add to the growing body of evidence that
ZDV readily induces genetic damage (mutational and clastogenic) in both nuclear and
mitochondrial DNA in a variety of in vitro and in vivo test systems (Sussman et al.,
1999;Poirier et al., 2004;Chan et al., in press;Von Tunglen et al., 2004;Meng et al.,
2002;Olivero et al., 2002;Bishop et al., 2004;Witt et al., 2004;Meng et al., 2000; Hong et al.,
in press; Olivero, in press).
Two observations may help to explain the seeming contradiction of the declining %MN-RET
observed in infants during postpartum ZDV prophylaxis. First, DNA incorporation of ZDV
and resulting genetic effects may be enhanced by the presence of additional nucleoside
analogues such as lamivudine or didanosine (Meng et al., 2002;Bishop et al., 2004;Witt et al,
2004;Meng et al., 2000), although neither of these two nucleosides alone induces micronuclei
in mice (Phillips et al., 1991;Von Tungeln et al., 2004;Witt et al., 2004;Von Tungeln et al.,
2002; Von Tungeln et al., in press). All women in this study who received ZDV during the
prenatal period also received lamivudine, whereas infants received ZDV alone during
postpartum treatment. Thus, transplacental exposure to multiple antiretroviral nucleosides may
have contributed to the high levels of micronucleated reticulocytes observed in neonates
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immediately after birth compared with the lower levels seen 4–6 weeks after birth. However,
it is more likely that the marked reduction in erythropoiesis that occurs in the newborn at birth
and persists for ~6–8 weeks (Palis and Segel, 1998) was largely responsible for the drop in
MN-RET frequencies observed during postpartum ZDV treatment. In the absence of cell
division, no induction of micronuclei can occur and, because the human spleen rapidly
sequesters and destroys damaged erythrocytes, the existing pool of micronucleated
reticulocytes would be quickly depleted. Indeed, flow cytometric determinations of
erythropoietic index showed marked decreases in most of the 4–6 week infant blood samples
(data not shown). Therefore, the declining MN-RET frequencies observed in the infants in this
study during ZDV postpartum treatment should not be interpreted as indicating reduced risk
to other exposed cell populations that continue to undergo rapid division in the neonate.
We recognize that our results are derived from a small number of study subjects, especially
those in the group that did not receive ZDV prenatally (only 3 subjects). However, the
consistency in response among both women and infants within the two subgroups (+ZDV,
−ZDV) provides strong evidence that prenatal exposure to ZDV is indeed genotoxic to
erythrocyte precursor cells, as evidenced by the markedly elevated frequencies of MN-
RETCD71+ observed in HIV-uninfected infants born to women who received ZDV-based
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prenatal ART. Furthermore, prenatal exposure to the nucleosides tenofovir, emtricitabine,
didanosine, or lamivudine, in the absence of ZDV, did not result in elevated frequencies of
MN-RET in the three infants or their mothers enrolled in this study.
Given the results of the study reported here, we recommend long-term monitoring of ZDV-
exposed HIV-uninfected infants to ensure their continued health. We must emphasize that we
do not advocate eliminating the use of ZDV in the treatment of HIV infection: ZDV-based
ART is highly effective in preventing mother-to-child transmission of a devastating disease.
However, we do hope that the results of this study will stimulate a search for and acceptance
of non-ZDV-based antiretroviral therapies that are equally effective in preventing maternal
transmission of the virus, while securing maternal health, but that have less potential for
genotoxicity in healthy non-infected infants.
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Acknowledgements
The authors are most grateful for the technical assistance provided by Ellen Dempsey, Kelley Montague, and Rosa
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Yeh, at the University of North Carolina Departments of Medicine and Pharmacy. At the Duke University Medical
Center, Department of Pediatric Infectious Disease, Margaret Donnelly, Felicia Wiley, Kareema Whitfield, Juliana
Simonetti, and Opemipo Johnson provided excellent support for this study. We thank Drs. John Bucher, Michael D.
Shelby, and June K. Dunnick, NIEHS, for their critical review of the manuscript.
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Fig 1.
%MN-RETCD71+ by time and prenatal ZDV exposure.
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