Molecular analysis of mutations at the HPRT and TK loci of human lymphoblastoid cells after combined treatments with 3′‐azido‐3′‐deoxythymidine and 2′,3′‐dideoxyinosine†

Abstract

Combinations of antiretroviral drugs that include nucleoside reverse transcriptase inhibitors (NRTIs) are superior to single-agent regimens in treating or preventing HIV infection, but the potential long-term health hazards of these treatments in humans are uncertain. In earlier studies, our group found that coexposure of TK6 human lymphoblastoid cells to 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), the first two NRTIs approved by the FDA as antiretroviral drugs, produced multiplicative synergistic enhancement of DNA incorporation of AZT and mutagenic responses in both the HPRT and TK reporter genes, as compared with single-drug exposures (Meng Q et al. [2000a]: Proc Natl Acad Sci USA 97:12667-12671). The purpose of the current study was to characterize the mutational specificity of equimolar mixtures of 100 microM or 300 microM AZT + ddI at the HPRT and TK loci of exposed cells vs. unexposed control cells, and to compare the resulting mutational spectra data to those previously found in cells exposed to AZT alone (Sussman H et al. [1999]: Mutat Res 429:249-259; Meng Q et al. [2000b]: Toxicol Sci 54:322-329). Molecular analyses of HPRT mutant clones were performed by reverse transcription-mediated production of cDNA, PCR amplification, and cDNA sequencing to define small DNA alterations, followed by multiplex PCR amplification of genomic DNA to define the fractions of deletion events. TK mutants with complete gene deletions were distinguished by Southern blot analysis. The observed HPRT mutational categories included point mutations, microinsertions/microdeletions, splicing-error mutations, and macrodeletions including partial and complete gene deletions. The only significant difference or shift in the mutational spectra for NRTI-treated cells vs. control cells was the increase in the frequency of complete TK gene deletions following exposures (for 3 days) to 300 microM AZT-ddI (P = 0.034, chi-square test of homogeneity); however, statistical analyses comparing the observed mutant fraction values (measured mutant frequency x percent of a class of mutation) between control and NRTI-treated cells for each class of mutation showed that the occurrences of complete gene deletions of both HPRT and TK were significantly elevated over background values (0.34 x 10(-6) in HPRT and 6.0 x 10(-6) in TK) at exposure levels of 100 microM AZT-ddI (i.e., 1.94 x 10(-6) in HPRT and 18.6 x 10(-6) in TK) and 300 microM AZT-ddI (i.e., 5.6 x 10(-6) in HPRT and 34.6 x 10(-6) in TK) (P < 0.05, Mann-Whitney U-statistic). These treatment-related increases in complete gene deletions were consistent with the spectra data for AZT alone (ibid.) and with the known mode of action of AZT and ddI as DNA chain terminators. In addition, cotreatments of ddI with AZT led to substantial absolute increases in the mutant fraction of other classes of mutations, unlike cells exposed solely to AZT [e.g., the frequency of point mutations among HPRT mutants was significantly increased by 130 and 323% over the background value (4.25 x ...

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

“…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). …”

“…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, 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, 1999Olivero 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, 2004Olivero et al, 2000Olivero et al, ,2002.…”

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

“…However, AZT also causes point mutations in human cells exposed in culture and in skin and lung neoplasms from in utero exposed mice [Zhang et al, 1998;Meng et al, 2002, Hong et al, 2007. The finding of increased GPA N/N Vfs in AZT-3TC exposed mother-child pairs is noteworthy because it reflects genetic events leading to LOH, a key mechanism directly relevant to NRTI-induced mutagenesis as shown by LOH in the thymidine kinase (TK) and adenine phosphoribosyltransferase (APRT) reporter genes in human lymphoblastoid cells exposed in vitro to AZT, or AZTdidanosine, and at the Tk locus of mice exposed in utero to AZT [Meng et al, 2000a[Meng et al, ,b,c, 2002Mittelstaedt et al, 2004;Von Tungeln et al, 2004.…”

Frequency of Tk and Hprt lymphocyte mutants and bone marrow micronuclei in mice treated neonatally with zidovudine and didanosine

“…ddI also appears to potentiate the mutagenicity of AZT because when human TK6 lymphoblastoid cells were incubated with AZT, ddI or a mixture of AZT and ddI, the combination of AZT and ddI increased the incorporation of AZT into cellular DNA and the mutant frequency within the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) genes (Meng et al, 2000). The increase in mutant frequency was attributed to an increase in point mutations (Meng et al, 2002).In previous work we demonstrated that AZT, but not the nucleoside analog lamivudine [(±)2¢,3¢-dideoxy-3¢-thiacytidine, 3TC], was genotoxic to neonatal mice, as indicated by an increased mutant frequency in the Tk gene and an increased frequency of micronucleated polychromatic erythrocytes (PCEs). Furthermore, 3TC in combination with AZT did not alter the responses observed with AZT alone (Von Tungeln et al, 2002).…”