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 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 mitochondrial DNA mutations (Chan et al., in press) and elevated mutation frequencies in lymphocytes (von Tungeln et al., 2002
; Torres et al., 2007
). Furthermore, the incidences of K-ras
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 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 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 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 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 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 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.