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Long-Term Mitochondrial Toxicity in HIV-Uninfected Infants Born to HIV-Infected Mothers
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JAIDS Journal of Acquired Immune Deficiency Syndromes June 2003; 33(2):175-183
Miriam C. Poirier; Rao L. Divi; Lena Al-Harthi; Ofelia A. Olivero; Vi Nguyen; Brettania Walker; Alan L. Landay; Vernon E. Walker; Manhattan Charurat; William A. Blattner; Women Infants Transmission Study (WITS) Group
Although children born to HIV-infected (HIV+) women receiving antiretroviral therapy during pregnancy show virtually no adverse clinical effects at birth, the antiretroviral nucleoside analog drugs are known to damage nuclear and mitochondrial DNA.
In this study, biomarkers of mitochondrial toxicity and genotoxicity have been examined in a well-characterized sample set consisting of infants born to HIV-uninfected (HIV-) mothers (n = 30), and HIV- infants (n = 20) born to HIV-infected (HIV+) mothers who received either no antiretroviral therapy (n = 10) or zidovudine (3`-azido-3`-deoxythymidine [AZT]) during pregnancy (n = 10). DNA from cord blood leukocytes and peripheral blood leukocytes taken at 1 and 2 years of age was examined for loss of mitochondrial DNA (mtDNA) and telomere integrity. Telomere length, a measure of nuclear DNA damage, was the same in all infants at birth and at age 1 year. The quantity of mtDNA was assessed relative to nuclear DNA using a polymerase chain reaction-based chemiluminescence detection (PCR-CID) method that determined mitochondrial D Loop gene copies relative to nuclear 18S RNA gene copies by comparison with a standard curve. MtDNA quantity was expressed as a ratio of gene copy numbers.
In infants of uninfected mothers (AZT-/HIV-) at the three time points, the ratios were 442 to 515, whereas in infants of untreated AZT-/HIV+ mothers the ratios were 261 to 297, and in infants of AZT-treated (AZT+/HIV+) mothers the ratios were 146 to 203. At all three time points, differences between the AZT-/HIV- group and the two HIV+ groups were statistically significant (p < .05), and differences between the AZT-/HIV+ and AZT+/HIV+ groups were also statistically significant (p < .05), demonstrating that AZT exposure causes a persistent depletion of mtDNA.
The study shows that children of HIV+ mothers are at risk for mitochondrial damage that is further increased in infants of mothers receiving AZT during pregnancy.
The authors stated that the results presented here underscore the necessity for long-term follow-up of children of HIV-infected mothers receiving prenatal HAART therapy, because it is possible that the depletion in mtDNA levels observed here may compromise their overall health later in life.
BACKGROUND: The current spread of HIV to the heterosexual population in the United States has resulted in approximately 7,000 children born yearly to HIV-infected mothers. When given during pregnancy, the antiretroviral nucleoside analog zidovudine (3`-azido-3`-deoxythymidine [AZT]) significantly reduces maternal-fetal viral transmission and thereby protects more than 1,000 children per year in the United States. The Centers for Disease Control's recommended treatment (from Protocol ACTG076 and http://www.hivatis. org/guideline/perinatal) is AZT given for the last 6 months of pregnancy, intravenous AZT dosing during labor and delivery, and 6 weeks of AZT administration to the infant after birth. In current clinical practice, the widely used drug combivir plus lamivudine, which is a second nucleoside analog (3`-dideoxy-3`-thiacytidine [3TC]), is more effective than AZT alone in inhibiting maternal-fetal HIV transmission. A number of studies have concluded that there are no persistent clinical changes in nucleoside analog-exposed infants compared with unexposed infants. The studies presented here were performed because mitochondrial compromise has been observed in some children exposed transplacentally to AZT (9) and telomere length shortening was observed in newborn mice transplacentally exposed to tumorigenic levels of AZT.
Long-term AZT use in adults has been associated with mitochondrial toxicity that is manifested in skeletal muscle and cardiac wasting, elevated serum lactic acid (15, 16), abnormal oxidative phosphorylation (OXPHOS)
enzyme-specific activities, depletion in the quantity of mitochondrial DNA (mtDNA), and morphologically damaged mitochondria with disrupted cristae and vacuolization. Until recently, similar nucleoside analog-induced mitochondrial toxicity was thought not to occur in HIV-uninfected infants born to HIV-infected mothers. However, Blanche et al. reported several nucleoside analog-exposed children between the ages of 7 months and 4.4 years with clinical mitochondrial dysfunction. The same study reported the deaths of two
children, at 11 and 13 months of age, with severe persistent mitochondrial dysfunction. In contrast, a retrospective analysis of infant mortality in several clinical trials (22) and a study of echocardiograms in exposed HIV-uninfected
infants (23) did not provide evidence of clinical mitochondrial toxicology after in utero exposure to AZT and/or 3TC. Further investigation will be required to resolve the apparent controversy.
Several studies have demonstrated that AZT targets telomeric DNA. Preferential incorporation of AZT and subsequent shortening of telomeric DNA occur in AZT-exposed cultured cells. The tumorigenic properties of AZT in a
transplacental mouse model have been described, and newborn mice in the same study had AZT incorporated into DNA and shortened telomeres in many of the same organs from which tumors arose. If telomere length is altered in DNA of human infants exposed to AZT, then there may be long-term consequences related to lifespan or other functions.
The Women and Infants Transmission Study (WITS), an ongoing, multi-center, prospective, cohort investigation of HIV-infected women and their infants, provides a well-characterized set of biological samples. The current analysis
used DNA from infant cord blood leukocytes and peripheral blood leukocytes taken at ages 1 year and 2 years from HIV-uninfected (HIV-) infants of HIV-infected (HIV+) women, all of which were supplied by the WITS. The samples
were obtained between 1990 and 1995, spanning a time when many HIV-infected women did not receive AZT or other antiretroviral therapy. Using the WITS leukocyte samples and age-matched leukocytes from HIV- pregnancies, we have selected a panel of biomarkers to investigate toxicities known to be associated with antiretroviral nucleoside analog use. The study included three groups: infants born to uninfected mothers (AZT-/HIV-), infants born to HIV+ mothers receiving no antiretroviral drugs (AZT-/HIV+), and infants born to HIV+ women receiving AZT (AZT+/HIV+). All samples were examined for mtDNA quantity and telomere length.
DISCUSSION by authors
In this study we have examined infant leukocytes for biomarkers related to mitochondrial integrity (mtDNA quantity) and telomere length in HIV-uninfected infants at birth and ages 1 year and 2 years. These studies were performed because mitochondrial integrity can be compromised in adults given long-term AZT exposure and in children transplacentally exposed to AZT, and telomeres were shortened in newborn mice given transplacental exposure to
tumorigenic levels of AZT (10). For this study, samples were obtained from infants of HIV- women and infants of HIV+ women who received either no antiretroviral drugs or AZT during pregnancy. The data showed that compared to the AZT-/HIV- group, newborn children of the AZT-/HIV+ group had significant reductions in mtDNA levels that were persistent up to age 2 years. In addition, mitochondrial integrity was altered further with the use of AZT during
pregnancy, as a significant depletion of mtDNA levels was observed at birth and at ages 1 and 2 years in the AZT+/HIV+ group compared to the AZT-/HIV+ group. Telomere length, measured at birth and age 1 year, was not altered in relation to the HIV status of the mother or AZT exposure.
Most antiretroviral nucleoside analog drugs are dideoxy-type nucleosides that exert their antiretroviral properties after phosphorylation and incorporation into viral DNA by causing both termination of DNA chain replication and inhibition of the viral reverse transcriptase. These drugs become incorporated into both nuclear and mtDNA of the host and specifically inhibit the mitochondrial polymerase [gamma]. They also produce a truncation of mtDNA
replication and a depletion of mtDNA quantity. Evidence of mitochondrial dysfunction in HIV- infants exposed perinatally to nucleoside analog drugs is rare, because most drug-exposed HIV-uninfected children are clinically asymptomatic . However, Blanche et al. reported the deaths of two HIV-uninfected children of HIV-infected mothers with severe mitochondrial toxicity. These children were exposed transplacentally to AZT and 3TC and died at approximately 1 year of age. Blanche et al. also described six other infants exposed in utero to AZT and younger than age 5 years who had persistent clinical and biochemical manifestations of mitochondrial dysfunction that
included seizures, pathological NMR imaging, altered OXPHOS enzyme activities, and elevated lactate levels. A retrospective analysis of mortality in several cohorts of HIV-infected mothers and their infants by the Perinatal Safety Review Working Group and a study of echocardiograms in AZT-exposed infants by Lipschultz et al. showed a lack of clinical evidence of mitochondrial dysfunction. However, these studies did not include specific biochemical
testing for mitochondrial function as performed by Blanche et al.
Our study provides a clear demonstration of mtDNA damage, present at birth and persisting up to age 2 years, in clinically asymptomatic HIV- children of HIV+ mothers taking AZT. We have demonstrated that both the presence of HIV infection in the mother and AZT exposure appear to be independently associated with depleted mtDNA in infant leukocytes. In addition, the mtDNA depletion was persistent up to age 2 years, an observation that might elucidate the persistent mitochondrial dysfunction observed by Blanche et al. in children up to age 4 years. It is difficult to know how this level of mtDNA depletion translates into immediate clinical consequences, but apparently many newborn children experience some degree of mitochondrial damage that may persist for at least 2 years. The findings reported here for AZT alone are supported by a significant mtDNA depletion observed in cord blood DNA samples obtained more recently from infants of HIV+ mothers receiving combivir during pregnancy. Not only was there significant quantitative depletion of mtDNA levels but also
mitochondrial morphological damage was observed in umbilical cord samples of the same infants by electron microscopy.
The determination of mtDNA quantity was accomplished using the highly sensitive PCR-CID. Initial experiments using the conventional Southern blot methodology were not reproducible and failed to distinguish subtle changes in mtDNA quantity. The new method requires only nanogram quantities of cellular DNA, which makes it a potentially attractive method for use in the clinic. A quantitative PCR method that has some similarities to the PCR-CID has been used by Cote et al. who demonstrated a 44% reduction in mtDNA quantity in therapy-naive HIV-infected adult patients and a partially reversible further reduction of 78% in patients receiving HAART therapy while showing symptoms of hyperlactatemia. In addition, Cherry et al. used quantitative real-time PCR to demonstrate a statistically significant 20% reduction in mtDNA level in thigh fat from HIV+ adult patients taking antiretroviral nucleoside analog drugs
compared with HIV- adults. The development and widespread use of these new highly sensitive methods for measuring mtDNA depletion may facilitate the early prediction of symptoms of mitochondrial toxicity before the onset of severe manifestations, such as lactic acidosis.
To date there is no epidemiologic evidence that in utero AZT exposure might be tumorigenic in children. Hanson et al. investigated 727 children with known AZT exposure enrolled in the PACTG 076 protocol, the PACTG 219 protocol, and the WITS. It was concluded that in children younger than age 5 years, there was no increased cancer risk caused by in utero drug exposure. Although this is reassuring, chemically induced tumors take 20 to 30 years to develop
and the possibility still exists that exposed children might have an elevated cancer risk that will be manifested later in life. Because AZT has been demonstrated to incorporate into cord blood leukocyte DNA and because there is evidence that children of women taking combivir are born with an elevated mutation rate , these children are clearly subject to antiretroviral nucleoside analog-induced genotoxicity. Another potential biomarker for genotoxic damage is shortened telomeres. In offspring of pregnant mice given AZT during gestation, shortened telomeres were observed in some organs. Also, while fetal monkeys exposed to a human-equivalent dose of AZT alone showed normal-length telomeres in virtually all organs, fetal monkeys exposed to the combination of AZT plus 3TC had telomere shortening in eight to nine different organs. The data in this study show that neither the AZT exposure nor the viral infection alters telomere length in infants up to age 1 year.
In general, HIV- children of HIV+ mothers taking antiretroviral therapy are born with no clinically evident abnormalities. However, the studies presented here provide evidence that infants of HIV+ women have depletions in mtDNA
quantity that persist up to age 2 years. Infants of HIV+ mothers receiving AZT exhibited a further significant depletion in mtDNA, which is not surprising given the mitochondrial toxicity in adult patients receiving antiretroviral nucleoside analog drugs and the mitochondrial dysfunction in some infants of HIV-infected women. In this study there was a reassuring lack of telomere shortening at birth and at age 1 year in children exposed to AZT. The use of antiretroviral drugs for the prevention of maternal-fetal viral transmission is widespread and highly successful. These drugs must be administered because current protocols save the lives of thousands of children each year. However, the results presented here underscore the necessity for long-term follow-up of children of HIV-infected mothers receiving prenatal HAART therapy, because it is possible that the depletion in mtDNA levels observed here may compromise their
overall health later in life.
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