Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Antiretroviral therapies based on nucleoside reverse transcriptase inhibitors (NRTIs), like zidovudine (3'-azido-3'-deoxythymidine; AZT) and lamivudine ((-)2',3'-dideoxy-3'-thiacytidine; 3TC), markedly reduce mother-to-child transmission of the human immunodeficiency virus (HIV). However, AZT induces damage in nuclear DNA of mice exposed in utero and postnatally, and mitochondrial DNA (mtDNA) damage has been observed in both human and mouse neonates following perinatal exposure to AZT and AZT/3TC in combination. To provide animal data modeling the NRTI-induced heart damage reported in human infants, we treated pregnant CD-1 mice throughout gestation and treated their pups by direct gavage from postnatal day (PND) 4 through PND 28 with daily doses of 150 mg/kg body weight (bw)/day AZT, 75 mg/kg bw/day 3TC, 125/62.5 mg/kg bw/day AZT/3TC, or the vehicle control. Half the pups were euthanized on PND 28; the remainder received no further dosing, and were euthanized at week 10. Heart tissue was collected, total DNA was extracted, and mtDNA copy number relative to nuclear DNA copy number, mtDNA damage, and mtDNA mutation assays were performed using PCR-based methods. Analyses revealed increases in mtDNA lesions in 4-week-old males and females treated with AZT or 3TC, but not in 10-week-old mice, suggesting that the damage resolved after treatment ceased. Interestingly, 10-week-old females treated with AZT/3TC had significant increases in mtDNA damage. Point mutations were elevated in 10-week-old females treated with AZT or AZT/3TC, but not 3TC; no increases in mutations were seen in either gender at 4 weeks of age. Our data suggest that AZT/3TC combination treatment produces greater mtDNA damage than either agent individually, and that female mice are more sensitive than males to AZT/3TC-induced mtDNA damage.
Environ Mol Mutagen
PMID:Mitochondrial toxicity in hearts of CD-1 mice following perinatal exposure to AZT, 3TC, or AZT/3TC in combination. 1639 92

Nucleoside analogs were first approved by the U.S. Food and Drug Administration for use against HIV-AIDS in 1987. Since then, these agents, now commonly referred to as nucleoside reverse transcriptase inhibitors (NRTIs), have become essential components of the Highly Active Antiretroviral Therapy (HAART) drug combinations used for treatment of Human Immunodeficiency Virus-1 (HIV-1) infections. Their antiretroviral activity is likely two-fold: incorporation of the drug into viral DNA and inhibition of the viral reverse transcriptase. However, incorporation of the drug into host nuclear and mitochondrial DNA may be largely responsible for dose-limiting toxicities. Azidothymidine (AZT, 3'-azido-3'-deoxythymidine, zidovudine), the first NRTI approved for the therapy of HIV-1, is incorporated into DNA, causes mutations in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) and thymidine kinase (TK) genes, and induces micronuclei, chromosomal aberrations, sister chromatid exchange, shortened telomeres, and other genotoxic effects in cultured cells. Genomic instability would be predicted as a consequence of these events. Metabolic pathways that result in the phosphorylation of AZT play a crucial role in AZT-DNA incorporation, and may be altered after prolonged treatment. For example, thymidine kinase 1, the enzyme responsible for AZT mono-phosphorylation, is down-regulated during long-term exposure and appears to be associated with AZT-induced replication inhibition and the accumulation of cells in S-phase. Detailed information on the mechanisms underlying NRTI-associated antiretroviral efficacy, toxicity, and metabolic resistance were not available when AZT was first approved for use as an antiretroviral agent. Current insights, based on 15 years of research, may lead to intervention strategies to attenuate toxicity without altering drug efficacy.
Environ Mol Mutagen
PMID:Mechanisms of genotoxicity of nucleoside reverse transcriptase inhibitors. 1639 95

We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship (QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between -30 and -150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K0.5 of 8.5+/-0.3 mM for Na+ and a Hill coefficient of 2.2+/-0.25 independent of membrane potential. We measured the Imax and K0.5 for substrate at -50 mV for the nucleoside analog drugs gemcitabine (638+/-58 nA, 59.7+/-17.5 microM), ribavirin (546+/-37 nA, 61.0+/-13.2 microM), AZT (420+/-4 nA, 310+/-9 microM), and 3-deazauridine (506+/-30 nA, 50.8+/-9.90 microM). K0.5 and Imax for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3'-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3-position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3'-OH and 5'-O and the hydrophobic center occupied by the base ring.
Mol Pharmacol 2006 May
PMID:Electrophysiological characterization and modeling of the structure activity relationship of the human concentrative nucleoside transporter 3 (hCNT3). 1644 84

Effective reduction in maternal-fetal human immunodeficiency virus-1 (HIV-1) transmission has been achieved by administration of nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy, and although most exposed children are clinically normal at birth, mitochondrial dysfunction has been reported. To examine mitochondrial integrity on a molecular level, we evaluated mitochondrial morphology by electron microscopy (EM) and mitochondrial DNA (mtDNA) quantity in umbilical cords and cord blood from NRTI-exposed and unexposed human and monkey newborns. Human subjects included infants born to HIV-1-infected mothers who received Combivir (Zidovudine [AZT] plus Lamivudine [3TC]) (n = 9) or AZT plus Didanosine [ddI] (n = 2) during pregnancy, and infants born to HIV-1-uninfected mothers (n = 7). NRTI-exposed Erythrocebus patas monkey dams (n = 3 per treatment group) were given human-equivalent dosing regimens containing 3TC, AZT/3TC, AZT/ddI, or Stavudine (d4T)/3TC during gestation. Four infants born to unexposed patas dams served as controls. Mitochondria in umbilical cord endothelial cells from NRTI-exposed monkey and human infants showed substantial abnormal pathology by EM, the extent of which was quantified from coded photomicrographs and shown to be different (P < 0.05) from the unexposed monkey and human newborns. Significant (P < 0.05) mtDNA depletion was found in umbilical cords from both human and monkey NRTI-exposed infants and in human, but not in monkey, cord blood leukocytes. For umbilical cords, an increase in mitochondrial morphological damage correlated with reduction in mtDNA quantity in fetal monkeys (r = 0.94). The treatment-induced mitochondrial compromise in infant monkeys ranked as follows: d4T/3TC > AZT/ddI > AZT/3TC > 3TC. The study demonstrates that transplacental NRTI exposures induce similar mitochondrial damage in cord blood and umbilical cords taken from retroviral-uninfected monkey infants and from human infants born to HIV-1-infected women.
Environ Mol Mutagen
PMID:Transplacentally exposed human and monkey newborn infants show similar evidence of nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity. 1653 87

In previous studies, we have shown that zidovudine (3'-azido-3'-deoxythymidine; AZT), but not lamivudine [(-)2',3'-dideoxy-3'-thiacytidine; 3TC], is genotoxic when administered to neonatal mice, and that 3TC when coadministered with AZT does not alter the responses observed with AZT alone (Von Tungeln et al. [2002] Carcinogenesis 23:1427-1432). We now have investigated the transplacental transfer of these drugs and the induction of mutants and micronuclei in the neonatal offspring. From gestational day 12 until parturition, female C57BL/6N and C57BL/6N/Tk(+/-) mice, which had been mated to male C3H/HeNMTV mice, were treated daily by gavage with AZT, 3TC, or a combination of AZT and 3TC. In both dams and fetuses, AZT was found at much higher levels than its metabolites, AZT 5'-glucuronide and 3'-azido-3'-deoxythymidine. In the neonates, AZT and the mixture of AZT and 3TC caused a decrease in the percentage of reticulocytes (RETs) and an increase in the percentage of micronucleated RETs and micronucleated normochromatic erythrocytes. When assessed 3 weeks after birth, AZT and the combination of AZT and 3TC increased the thymidine kinase (Tk) mutant frequency in male mice; at 5 weeks, 3TC increased the Tk mutant frequency in female mice. The increase in Tk mutants in mice treated with AZT and the mixture of AZT and 3TC was associated with loss of the wild-type (Tk(+)) allele (loss of heterozygosity; LOH) and a pattern of discontinuous LOH. These data indicate that AZT, 3TC, and the combination of AZT and 3TC are transplacental mutagens and that the increase in mutants resulting from AZT is due mainly to large-scale genetic alterations.
Environ Mol Mutagen
PMID:Transplacental drug transfer and frequency of Tk and Hprt lymphocyte mutants and peripheral blood micronuclei in mice treated transplacentally with zidovudine and lamivudine. 1685 Apr 53

Long-term use of antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) as therapy for human immunodeficiency virus-1 (HIV-1) infection is limited by mitochondrial toxicity. Here we document mitochondrial pathology during the long-term culture of human HeLa cells in the presence or absence of the NRTI Zidovudine(R) (AZT, 800 muM) for up to 77-passages (p), with samples taken at early (p5-p11), middle (p36 and p37), and late (p70-p77) passages. Samples were analyzed for changes in mitochondrial morphology, mitochondrial (mt)DNA quantity, nuclear and mitochondrial gene expression, and mitochondrial membrane potential. Mitochondria showed abnormal proliferation at p5 and abnormal morphology >/=p36. mtDNA quantity was increased at p5 and p11, and 65% depleted at p71. Hierarchical clustering of nuclear gene expression, examined at p37 by the NCI cDNA microarray in AZT-exposed cells, showed down-regulation of 13 out of 16 lipid-metabolizing genes, and up-regulation of most oxidative phosphorylation (OXPHOS) genes. OXPHOS genes encoded by mtDNA, examined at p5, p36, and p75 using the Mitochondrial Gene Mini Array, revealed up-regulation of genes coding for polypeptides of NADH dehydrogenase, ATP synthase, and cytochrome c oxidase. Mitochondrial membrane potential, monitored by JC1 staining, was elevated at p10 and p32, and essentially completely absent at p71. The data show that during chronic exposure of HeLa cells to AZT, a compensatory response was induced at the earlier passages (p5-p37), and by p71 there was widespread mitochondrial morphological damage, severe mtDNA depletion, and a substantial loss of mitochondrial membrane potential.
Environ Mol Mutagen
PMID:Morphological and molecular course of mitochondrial pathology in cultured human cells exposed long-term to Zidovudine. 1689 29

Human immunodeficiency virus type 1 (HIV-1) strains having dipeptide insertions in the fingers subdomain and other drug resistance-related mutations scattered throughout their reverse transcriptase (RT)-coding region show high-level resistance to zidovudine (AZT) and other nucleoside analogues. Those phenotypic effects have been correlated with their increased ATP-dependent phosphorolytic activity on chain-terminated primers. Mutations T69S and T215Y and a dipeptide insertion (i.e. Ser-Ser) between positions 69 and 70 are required to achieve low-level resistance to thymidine analogues. However, additional amino acid substitutions are necessary to achieve the high-level phenotypic resistance to AZT shown by clinical HIV isolates carrying a dipeptide insertion in their RT-coding region. In order to identify those mutations that contribute to resistance in the sequence context of an insertion-containing RT derived from an HIV clinical isolate (designated as SS RT), we expressed and purified a series of chimeric enzymes containing portions of the wild-type or SS RT sequences. ATP-mediated excision activity measurements using AZT- and stavudine (d4T)-terminated primers and phenotypic assays showed that molecular determinants of high-level resistance to AZT were located in the fingers subdomain of the polymerase. Further studies, using recombinant RTs obtained by site-directed mutagenesis, revealed that M41L, A62V and in a lesser extent K70R, were the key mutations that together with T69S, T215Y and the dipeptide insertion conferred high levels of ATP-dependent phosphorolytic activity on AZT and d4T-terminated primers. Excision activity correlated well with AZT susceptibility measurements, and was consistent with phenotypic resistance to d4T. Structural analysis of the location of the implicated amino acid substitutions revealed a coordinated effect of M41L and A62V on the positioning of the beta3-beta4 hairpin loop, which plays a key role in the resistance mechanism.
J Mol Biol 2007 Jan 12
PMID:Mutational patterns associated with the 69 insertion complex in multi-drug-resistant HIV-1 reverse transcriptase that confer increased excision activity and high-level resistance to zidovudine. 1707 May 43

The prophylactic use of zidovudine (3'-azido-3'-deoxythymidine, AZT) during pregnancy greatly reduces transmission of HIV-1 from infected mothers to their infants; however, the affinity of host cell DNA polymerases for AZT also allows for its incorporation into host cell DNA, predisposing to cancer development. To expand upon previous transplacental carcinogenesis assays performed in CD-1 mice, the transplacental carcinogenicity of AZT was evaluated in a second mouse strain and a second rodent species. Date-mated female mice and rats were gavaged daily with 0, 80, 240, or 480 mg AZT/kg bw during the last 7 days of gestation. At 2 years postpartum, male and female B6C3F1 mouse and F344 rat offspring (n = 44-46 of each sex and species/treatment group) were necropsied for gross and microscopic tissue examinations. Under the conditions of these two-year studies, there was clear evidence of carcinogenic activity based upon significant dose-related trends and increases in the incidences of hemangiosarcoma in male mice and mononuclear cell leukemia in female rats. There was some evidence of carcinogenic activity in the livers of male mice based upon a positive trend and an increased incidence of hepatic carcinoma in the high-dose AZT group. The incidence of gliomas in female rats exceeded the historical background rates for gliomas in F344 rats. P53 overexpression was detected in some AZT-treated mouse neoplasms. These and other cancer-related findings confirm and extend those of previous transplacental carcinogenicity studies of AZT in mice, support the need for long-term follow-up of nucleoside reverse transcriptase inhibitor (NRTI)-exposed children, and indicate the necessity for effective protective strategies against NRTI-induced side effects.
Environ Mol Mutagen
PMID:Transplacental carcinogenicity of 3'-azido-3'-deoxythymidine in B6C3F1 mice and F344 rats. 1735 26

The genotoxicity of zidovudine (AZT) based treatments was investigated in human H9 lymphoblastoid cells in an in vitro study and in red blood cells (RBCs) from perinatally exposed HIV-1-infected mothers and their infants in an observational cohort study. Exposure of H9 cells for 24 hr to AZT produced dose-dependent increases in Comet assay tail moment (TM) when electrophoresed at pH 13.0, but not at pH 12.1 or pH 8.0, suggesting that DNA damage was via alkali-labile lesions and not double-stranded DNA strand breaks. The TM dose response at pH 13.0 correlated directly with AZT-DNA incorporation determined by AZT-radioimmunoassay. Levels of DNA damage in utero, measured by Comet assay TM, were similar in cord blood mononuclear cells of nucleoside analog-exposed newborns (n = 43) and unexposed controls (n = 40). In contrast, the glycophorin A (GPA) somatic cell mutation assay (which screens for large-scale DNA damage in RBCs) showed clear evidence that GPA N/N variants, arising from chromosome loss and duplication, somatic recombination, and gene conversion, were significantly elevated in mother-child pairs receiving prepartum AZT plus lamivudine (3TC). Cord blood from newborns exposed to AZT-3TC had GPA N/N variant frequencies of 4.7 +/- 0.7 (mean +/- SE) x 10(-6) RBCs (n = 26 infants) compared with 2.2 +/- 0.3 x 10(-6) RBCs for unexposed controls (n = 30 infants; P < 0.001). Elevations in GPA N/N variants generally persisted through 1 year of age in nucleoside analog-exposed children. Overall, the mutagenic effects found in mother-child pairs receiving AZT-based treatments justify their surveillance for long-term genotoxic consequences.
Environ Mol Mutagen
PMID: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. 1735 27

Antiretroviral therapy is highly effective in reducing vertical transfer of HIV infection, sparing many thousands of children premature death from AIDS. However, accumulating evidence indicates that perinatal exposure to antiretroviral agents may place them at elevated risk of developing cancer later in life, owing to potential carcinogenic effects of the agents. An initial experimental evaluation clearly demonstrated that AZT was a genotoxin and transplacental carcinogen of intermediate potency in CD-1 mice. This issue of Environmental and Molecular Mutagenesis contains reports of recent studies designed to confirm and extend earlier findings, and to provide further perspective that will facilitate development of strategies through which the adverse effects might be mitigated. The studies focused on various aspects of the genotoxicity and carcinogenicity of antiretroviral agents, including: mutagenesis in several in vitro experimental systems; mutations and clastogenic effects induced by transplacental administration in mice; transplacental carcinogenesis and mutations in oncogenes and tumor suppressor genes in tumors of mice; and genotoxicity and clastogenicity following perinatal exposure of HIV-infected mothers and their uninfected infants. Collectively, the results obtained provide convincing biological plausibility for the postulate that perinatal exposure to nucleoside analogs puts children at elevated risk of developing cancers later in life. They further emphasize the importance of continued surveillance of these children for increased cancer risk and indicate a need for efforts to develop less genotoxic alternative agents.
Environ Mol Mutagen
PMID:Does perinatal antiretroviral therapy create an iatrogenic cancer risk? 1735 28


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