J Infect Dis. 2013 Jul 15;208(2):244-8. doi: 10.1093/infdis/jit146.

Perinatal exposure of patas monkeys to antiretroviral nucleoside reverse trans-criptase inhibitors (NRTIs) induces genotoxicity persistent for up to 3 years of age.

Ofelia A. Olivero1, Lorangelly Rivera Torres1, Sayeh Gorjifard1, Dariya Momot1, Eryney Marrogi1, Rao L. Divi1,2, Yongmin Liu1, Ruth A. Woodward3, Marsha J. Sowers3 and Miriam C. Poirier1

1 Carcinogen-DNA Interactions Section, Laboratory of Cancer Biology and Genetics, CCR, National Cancer Institute, NIH, 37 Convent Drive, Bethesda, MD 20892-4255.

Methods and Technologies Branch, Division of Cancer Control and Population Sciences, National Cancer Institute, NIH, Executive Plaza North, Bethesda, MD, 20892-7324.

3 NIH Animal Center, Shared Animal Faclitiy,16701 Elmer School Rd., Dickerson, MD, 20837.

 

ABSTRACT

Antiretroviral NRTIs are given during pregnancy and after birth to reduce maternal-fetal HIV-1 transmission. These drugs are DNA replication chain terminators, and induce: centrosomal amplification (CA, >2 centrosomes/nucleus), resulting in abnormal chromosomal segregation; micronuclei (MN); and, MN containing whole chromosomes (MN+C).  Pregnant Erythrocebus patas dams (n=2-4/group) were given human-equivalent daily NRTI dosing with AZT/3TC, AZT/3TC/Abacavir (ABC), or AZT/3TC plus the non-NRTI Nevirapine (NVP), for the last half (10 wk) of gestation.  Patas offspring taken at 1 and 3 yr were also dosed for 6 wk post-birth.  Mesenchymal fibroblasts obtained from bone marrow of patas at birth, 1 and 3 yr of age, were grown to confluence in culture, and with no other treatment, were examined for CA, MN, and MN+C.  Compared to controls, significant increases in CA, MN and MN+C were found in offspring exposed in utero to AZT/3TC and examined at birth, and offspring exposed in utero to AZT/3TC/ABC and examined at 1 year (p<0.05).  Offspring examined at 3 yr, after exposure to AZT/3TC/NVP, showed increased MN and MN+C (p<0.05), while those exposed to AZT/3TC/ABC had increased CA and MN+C (p<0.05).  These genotoxic end points reflect spindle malformations (CA and MN), as well as chromosome loss (MN), and loss of a whole chromosome leading to aneuploidy (MN+C).  The study shows that abnormal chromosomal segregation and aneuploidy, initiated by in utero NRTI exposure, persist in patas bone marrow up to 3 years of age (15 yr human equivalent), suggesting that children born to HIV-1-infected mothers receiving NRTI therapy may sustain genotoxicity and be at increased risk for cancer.

 

Supplement

Antiretroviral (ARV) drug combinations used as therapy for inhibition of maternal-fetal vertical HIV-1 transmission provide a highly-successful approach for preventing the spread of HIV-1/AIDS (1, 2).  ARVs are typically given to HIV-1-infected pregnant women during part or all of pregnancy, labor and delivery, and to the infant during the first 6 weeks after birth.  Peripartum use of ARV drugs is successful in preventing mother-to-child transmission in ~ 99% of the 10,000 HIV-1-infected women who become pregnant yearly in the United States.  Most ARV drug combinations used in human pregnancy contain 2-3 NRTIs, and the drug Combivir©, a combination of the NRTIs Zidovudine (AZT) and Lamivudine (3TC), was widely used in pregnancy as the standard of care for many years.

Incorporation of NRTIs into RNA and DNA, followed by nucleic acid replication arrest, are essential for the success of HIV-1 therapy, however both incorporation and replication arrest also occur in host nuclear DNA (3), resulting in mutagenesis and the potential for cancer induction (Table 1). Transplacental studies, in which pregnant mice were exposed to AZT daily for the last week of gestation, showed incorporation of AZT into fetal organ DNA, as well as mutagenesis and tumor induction in the offspring (4-6).  In infant patas monkeys born to dams receiving human-equivalent NRTI protocols while pregnant, incorporation of AZT and 3TC into nuclear DNA was documented in many fetal organs (7).  Table 1, based on (3, 8), shows that the levels of AZT and 3TC incorporated into patas monkey organ DNA were similar to those found in human placental DNA, where HIV-1-infected mothers received AZT to prevent mother-to-child transmission, and in organs of mouse offspring exposed transplacentally to a tumorigenic dose of AZT.  Furthermore, levels of NRTI-induced mutagenicity (Table 1) in human infants exposed to AZT or AZT/3TC in utero (3, 8, 9), were similar to those found in the mice exposed transplacentally to a tumorigenic dosing regimen of AZT, suggesting the possibility of an increased risk for cancer induction children exposed to  NRTIs in utero (10) (Table 1).

In the study presented here (11) the ex vivo approach, revealed persistent genotoxicity in patas offspring up to 3 years of age after in utero and perinatal exposure to ARV combinations. This study was published with a companion paper by Andre-Schmutz et al. (12) who showed increased chromosomal aberrations in leukocytes taken at birth from human infants exposed in utero to AZT, compared to unexposed infants. Both studies showed AZT-induced aneuploidy in blood samples taken at birth from humans and patas, providing evidence for AZT-induced aneuploidy. In addition, because a three year old patas is equivalent in development to a 15 year old human, the patas, which had no ARV exposures past 6 wk of age, would appear to sustain persist long-term genotoxic damage in bone marrow stem cells.  This elucidates mechanisms underlying   AZT-induced transplacental tumorigenicity in mice, and suggests that children born to HIV-1-infected mothers receiving ARV therapy may be at increased risk of cancer.

 

Table 1 – Genotoxicity in Mouse, Monkey and Human Fetuses Exposed in utero to AZT or AZT/3TC   tab1* The mouse total dose, received during the last week of gestation, was 3.06 gm AZT/mouse and the human total, received by one mother during the last 6 months of gestation was 1.5 gm AZT.   **NA = no assay

 

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2.         Mofenson LM, McIntyre JA. Advances and research directions in the prevention of mother-to-child HIV-1 transmission. Lancet. 2000; 355(9222):2237-44.

3.         Poirier MC, Olivero OA, Walker DM, et al. Perinatal genotoxicity and carcinogenicity of anti-retroviral nucleoside analog drugs. Toxicol.Appl.Pharmacol. 2004; 199(2):151-61.

4.         Olivero OA, Anderson LM, Diwan BA, et al. Transplacental effects of 3′-azido-2′,3′-dideoxythymidine (AZT): tumorigenicity in mice and genotoxicity in mice and monkeys. J.Natl.Cancer Inst. 1997; 89:1602-8.

5.         Walker DM, Malarkey DE, Seilkop SK, et al. Transplacental carcinogenicity of 3′-azido-3′-deoxythymidine in B6C3F1 mice and F344 rats. Environ.Mol.Mutagen. 2007; 48(3-4):283-98.

6.         Dobrovolsky VN, Shaddock JG, Mittelstaedt RA, et al. Frequency of Hprt mutant lymphocytes and micronucleated erythrocytes in p53-haplodeficient mice treated perinatally with AZT and AZT in combination with 3TC. Environmental and molecular mutagenesis. 2007; 48(3-4):270-82. doi:10.1002/em.20280

7.         Olivero OA, Fernandez JJ, Antiochos BB, et al. Transplacental genotoxicity of combined antiretroviral nucleoside analogue therapy in Erythrocebus patas monkeys. J.Acquir.Immune.Defic.Syndr. 2002; 29(4):323-9.

8.         Escobar PA, Olivero OA, Wade NA, et al. Genotoxicity assessed by the comet and GPA assays following in vitro exposure of human lymphoblastoid cells (H9) or perinatal exposure of mother-child pairs to AZT or AZT-3TC. Environ.Mol.Mutagen. 2007; 48(3-4):330-43.

9.         Meng Q, Olivero OA, Fasco MJ, et al. Plasma and cellular markers of 3′-azido-3′-dideoxythymidine (AZT) metabolism as indicators of DNA damage in cord blood mononuclear cells from infants receiving prepartum NRTIs. Environ.Mol.Mutagen. 2007; 48(3-4):307-21.

10.       Benhammou V, Warszawski J, Bellec S, et al. Incidence of cancer in children perinatally exposed to nucleoside reverse transcriptase inhibitors. AIDS. 2008; 22(16):2165-77.

11.       Olivero OA, Torres LR, Gorjifard S, et al. Perinatal Exposure of Patas Monkeys to Antiretroviral Nucleoside Reverse-Transcriptase Inhibitors Induces Genotoxicity Persistent for up to 3 Years of Age. The Journal of infectious diseases. 2013; 208(2):244-8. doi:10.1093/infdis/jit146

12.       Andre-Schmutz I, Dal-Cortivo L, Six E, et al. Genotoxic signature in cord blood cells of newborns exposed in utero to a Zidovudine-based antiretroviral combination. The Journal of infectious diseases. 2013; 208(2):235-43. doi:10.1093/infdis/jit149

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