Nuclear bud formation: a novel manifestation of Zidovudine genotoxicity.

Normal diploid somatic mammalian cell division generates 2 daughter cells as a result of a strict and well-controlled mitotic process. However, some defects during the progression of that process could generate an unbalanced distribution of chromosomes, aneuploidy and eventually, a malignant phenotype. Previous observations using a transgenic mouse model with diminished DNA repair capacity revealed the presence of nuclear buds (NBs) induced in vitro by the nucleoside analog zidovudine (Retrovir(R), 3'-azido-3'-deoxythymidine, AZT). Here we used bone marrow mesenchymal cells, taken from mice with the Xpa(-/-)Trp53(+/-) genotype, that were cultured and exposed to 0 and 100 muM AZT for 24 hours. Fixed and denatured cells were processed by fluorescence in situ hybridization (FISH) with whole chromosome painting probes used to identify chromosomes in cells growing on glass chamber slides (2 probes/slide). A variety of sizes and shapes of NBs were observed. Some NBs had a large connection with the main nucleus (>(1/4) of the NB diameter), others hada smaller connection (<(1/4) of the NB diameter), some were circular and positioned close to the nucleus, while some resided in the cytoplasm separated from the nucleus or connected by a thin chromatin strand. We had hypothesized that NBs would progress in the process of budding until separation occurred, but this was not proven by time-lapse photography studies performed for 20 hours. From 1,126 cells scored in the unexposed cultures, 10.39 % of cells carried NBs, while from 1,108 cells scored in the AZT-exposed cultures 29.16% of cells carried NBs (p = 0.001). In AZT-exposed cells there were a total of 322 NBs scored; 46.6% or 150 NBs contained positive signals for one or both probes used, while 53% or 172 NBs had no probe signal. In addition, FISH analysis showed no preferential localization of any chromosome within the NBs. Among the NBs that carried no probe signal, the presence of positive signals with inversion of DAPI imaging demonstrated centromeric content. It has been hypothesized that NBs occur as a result of expulsion of amplified DNA from the main nucleus; however, this data demonstrates that NBs may contain any chromosome, suggesting that NBs do not consist of just amplified DNA.

Plasma drug levels compared with DNA incorporation of 3'-azido-3'-deoxythymidine (AZT) in adult cynomolgus (Macaca fascicularis) monkeys.

Zidovudine (3'-azido-3'-deoxythymidine, AZT), widely used for the therapy of the Human Immunodeficiency Virus-1 (HIV-1), is a nucleoside analog of thymidine that becomes phosphorylated and incorporated into nuclear and mitochondrial DNA. Levels of AZT incorporation into DNA of humans, monkeys, and mice are highly variable and suggest interindividual variability in phosphorylation pathways. In addition, studies in rhesus monkeys (1) have shown a lack of correlation between levels of unbound AZT in plasma and tissue AZT-DNA. However, the correlation between plasma AZT and tissue AZT-DNA has not been previously examined in the same primate. Here we examine the relationship between AZT-DNA incorporation in leukocytes and multiple organs, and levels of the drug circulating in plasma of adult female cynomolgus (Macaca fascicularis) monkeys. Three monkeys were dosed with 40.0 mg of AZT/day for 30 days by naso-gastric intubation. The average daily dose of 9.9 mg of AZT/kg/body wt was similar to the approximately 8.6 mg of AZT/kg/body wt (600 mg/day) given to adult HIV-1-infected patients. In all three monkeys, at the time of sampling, values for AZT concentrations in plasma were similar and values for AZT incorporation into leukocyte DNA (86.1, 100.0, and 114.1 molecules of AZT/10(6) nucleotides) were also similar. AZT-DNA incorporation was detected in liver, uterus, spleen, and kidney from the three AZT-exposed animals, with values for positive samples ranging from 5.8 to 97.4 molecules of AZT/10(6) nucleotides. In brain cortex and lung DNA from AZT-exposed animals, AZT incorporation was undetectable. The data suggest that organ-specific differences in AZT uptake and/or metabolism may contribute to AZT phosphorylation and subsequent drug incorporation into DNA. In addition, AZT-DNA levels in monkey organs were similar to or lower than values observed in peripheral leukocytes of adult AIDS patients.

Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells.

Normal cells in culture divide a certain amount of times and undergo a process termed replicative senescence. Telomere loss is thought to control entry into senescence. Activation of telomerase in tumors bypasses cellular senescence and is thus a requirement for tumor progression. We reported previously the preferential incorporation of 3'-azido-2', 3'-dideoxythymidine (AZT) in telomeric sequences of immortalized cells in culture. In this work, we have investigated the effects of chronic in vitro AZT exposure on F3II mouse mammary carcinoma cells. We demonstrate, for the first time, that AZT-treated tumor cells have a reduced tumorigenicity in syngeneic BALB/c mice. Tumor incidence was reduced and survival was prolonged in animals inoculated with AZT-treated cells when comparing with control counterparts. The number and size of spontaneous metastases were also decreased in animals inoculated with AZT-treated cells. In addition, we present evidence of morphological and biochemical signs of senescence, as shown by the staining for senescence associated beta-galactosidase activity, and induction of programmed cell death, as demonstrated by an increase of caspase-3 activity, in tumor cells exposed to AZT. These data indicate that chronic exposure of mammary carcinoma cells to AZT may be sufficient to induce a senescent phenotype and to reduce tumorigenicity.

Incorporation of zidovudine into cord blood DNA of infants and peripheral blood DNA of their HIV-1-positive mothers.

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) is a weak carcinogen in adult female mice and a moderately strong carcinogen in the offspring of female mice given the drug during gestation. In addition, incorporation of AZT into DNA was observed in multiple organs of transplacentally exposed newborn mice. Here we investigate the incorporation of AZT into peripheral leukocyte DNA of HIV-1-positive adult pregnant women given AZT for variable times during gestation and cord blood of infants exposed to AZT in utero. The length of treatment varied between 10 days and 9 months. High molecular weight DNA was extracted from maternal peripheral blood mononuclear cells (PBMC) and infant cord blood. A specific AZT-DNA radioimmunoassay was used to determine the amount of AZT incorporated into leukocyte DNA. Incorporation of AZT into DNA ranged up to 183.3 and 344.5 molecules of AZT/10(6) nucleotides in the mothers and infants, respectively, and was detected in about 70% of samples. Therefore, AZT-induced mutagenic events are possible in the majority of adults and infants. No correlation was found between level of incorporation and length of AZT treatment, suggesting that the differences observed among the individuals arise from variability in AZT metabolism. These data support previous observations that a high degree of inter-individual variability in AZT phosphorylation occurs in primates.

Zidovudine-didanosine coexposure potentiates DNA incorporation of zidovudine and mutagenesis in human cells.

Drug combinations that include nucleoside reverse transcriptase inhibitors (NRTIs) are remarkably effective in preventing maternal-viral transmission of HIV during pregnancy. However, there may be potential long-term risks for children exposed in utero. Examination of the genotoxic and mutagenic effects of two NRTIs, zidovudine [AZT (3'-azido-3'-deoxythymidine)] and didanosine [ddI (2',3'-dideoxyinosine)], in cultured human lymphoblastoid cells revealed multiplicative synergistic enhancement of AZT-DNA incorporation and mutant frequency induction in response to the combined drug exposure, as compared with single-drug exposures. Dose-related increases in DNA incorporation of AZT (as measured by a competitive RIA) and mutagenicity at the HPRT and TK loci (as assessed by cell-cloning assays) were observed in cells exposed in culture to AZT, or equimolar combinations of AZT + ddI, at exposure concentrations ranging from 3 to 30 times the maximum plasma levels found in humans. Because mutagenesis is strongly associated with tumor induction in experimental models, children exposed transplacentally to combinations of NRTIs may be at risk for cancer development later in life.

Association between GSTM1*0 and squamous dysplasia of the esophagus in the high risk region of Linxian, China.

Individuals with specific phase I and phase II enzyme polymorphisms may be at increased risk for squamous cell carcinoma of the esophagus. However, to our knowledge there has been only one previous report that evaluates a potential role for these polymorphisms in increasing risk for preneoplastic squamous lesions of the esophagus. To explore this further, we examined polymorphisms in CYP1A1, CYP2E1, GSTM1 and GSTT1, both independently and in combination, for potential associations with the risk of biopsy-proven squamous dysplasia of the esophagus in asymptomatic adults from Linxian, a high risk region in China. Cases consisted of 56 individuals from an esophageal cancer screening study with an endoscopic biopsy diagnosis of mild or moderate squamous dysplasia. Each case was matched on age (+/- 1 year) and gender to a control. Controls were defined as screening study participants with an endoscopic biopsy diagnosis of normal mucosa or esophagitis. DNA was extracted from frozen cell samples obtained by cytologic balloon examination and genotyped using standard methods. Individuals who were GSTM1 null (homozygous for GSTM1*0) were found to have a tendency for an increased risk of esophageal squamous dysplasia (odds ratio=2.6, 95% CI, 0.9-7.4). No excess risks were observed for inheritance of other putative at risk genotypes CYP1A1*2B, CYP2E1*6 or GSTT1*0. The risk associated with the inheritance of combined genotypes was not significantly different than the risk estimates from the univariate analysis. These results are consistent with the notion that exposure to environmental carcinogens that are detoxified by GSTM1, such as polycyclic aromatic hydrocarbons, may contribute to the etiology of esophageal cancer in Linxian.

Absence of structural or functional alterations in male and female reproductive organs of F1 and F2 generations derived from female mice exposed to 3'-azido-3'-deoxythymidine during pregnancy.

To investigate the effects of in utero exposure to 3'-azido-3'-deoxythymidine (AZT) on male and female reproductive system development, pregnant CD-1 mice were given daily intragastric doses of 25.0 mg AZT during days 12 through 18 of gestation. The offspring were examined at birth, as well as at pubertal, young adult and adult stages of development, for reproductive organ endpoints including anogenital distance, onset of testicular descent, latency to vaginal opening, and proportion of time for each of the stages of estrous cycle. These reproductive endpoints remained mostly unchanged in AZT-treated offspring as compared to the controls. Males and females exposed in utero to AZT (F1 generation) were fertile when mated to untreated females and males, respectively, and their liveborn F2 offspring showed no adverse effects for any of the reproductive parameters tested. Thus, no evidence of developmental reproductive toxicity was noted either in the F1 mice exposed to AZT during the critical period of male and female reproductive system development, or in the F2 mice born of matings between the AZT-exposed F1 mice and unexposed animals.

Oxidative DNA damage in fetal tissues after transplacental exposure to 3'-azido-3'-deoxythymidine (AZT).

The nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) has been used successfully to reduce the incidence of transplacental and perinatal transmission of the HIV virus. However, prolonged treatment with high doses of AZT is utilized in this therapy, and AZT has been found to be a perinatal carcinogen in mice. Any possible perinatal carcinogenic side effects in the human can best be managed if the mechanism is understood. AZT targets mitochondria and might cause increased intracellular production of reactive oxygen species (ROS). We tested whether transplacental AZT may cause oxidative damage in nuclear DNA of fetal tissues. CD-1 Swiss pregnant mice were treated with the transplacental carcinogenesis regimen (25 mg/day AZT, for gestation days 12-18) and tissues collected on the day of birth. Significant increases in 8-oxo-2'-deoxyguano- sine (8-oxo-dG) were found in the livers, a target tissue for transplacental carcinogenesis, and in the kidneys. A non-significant increase occurred in brain, with no change in lung. Tissues were also obtained from fetal patas monkeys (Erythrocebus patas), whose mothers had received 10 mg AZT/day during the last half of gestation. Although limited numbers of samples were available, possible increases in 8-oxo-dG were noted, relative to controls, for placenta and for fetal lung and brain (P = 0.055 for treatment-related increases in these tissues). These results suggest that an increase in reactive oxygen species could contribute to the mechanism of transplacental carcinogenesis by AZT in mice, and that this may also occur in primates.

Relationships between DNA incorporation, mutant frequency, and loss of heterozygosity at the TK locus in human lymphoblastoid cells exposed to 3'-azido-3'-deoxythymidine.

3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the treatment of AIDS patients and for prevention of the onset of AIDS in HIV-seropositive individuals, causes tumors in mice exposed as adults or in utero. The purpose of this study was to investigate the potential mechanisms of AZT mutagenicity and carcinogenicity by quantifying the incorporation of AZT into cellular DNA, measuring AZT-induced thymidine kinase (TK) mutant frequencies (Mfs), and determining the percentage of loss of heterozygosity (LOH) in spontaneous or AZT-induced TK mutants in the human lymphoblastoid cell line, TK6. Cells were exposed to 300 microM AZT for 0, 1, 3, or 6 days, or to 0, 33, 100, 300, or 900 microM AZT for 3 days (n = 5 flasks/group). The effects of exposure concentration on incorporation of AZT into cellular DNA were evaluated by an AZT radioimmunoassay, and the effects of duration and concentration of AZT exposure on the TK Mfs were assessed by a cell-cloning assay. AZT was incorporated into DNA in a dose-related manner at concentrations up to 300 microM, above which no further increase was observed. TK Mf increased with the extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.036, coefficient = 0.903) between AZT-DNA incorporation and AZT-induced TK Mfs, suggesting that AZT incorporation into cellular DNA has a direct role in the genotoxicity of AZT. Southern blot analyses indicated that 84% (6.2 x 10(-6)/7.4 x 10(-6)) of AZT-induced mutants were attributable to LOH, consistent with the known mechanism of AZT as a DNA chain terminator. Considering the importance of LOH in human carcinogenesis, AZT-induced LOH warrants further study.

Multiorgan transplacental and neonatal carcinogenicity of 3'-azido-3'-deoxythymidine in mice.

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is used successfully for reduction of perinatal viral transmission. However toxic side effects including carcinogenesis are possible. To test this, pregnant CD-1 Swiss mice were given 25.0 or 12.5 mg AZT on gestation days 12-18. Previously we reported an increase in lung, liver, and female reproductive system tumors in offspring euthanized at 1 year (Olivero et al., J. Natl. Cancer Inst. 89, 1602-1608, 1997). Findings for all remaining offspring up to 2 years old are reported here. AZT effects were most prominent in female offspring, with a significant threefold increase in lung tumors, a reduction in lymphoblastic and follicle center cell lymphomas, and a significant increase in histiocytic sarcomas (0 in controls, 3% after low-dose AZT, and 8% after high-dose AZT, p = 0.022). Dose-dependent incidences of mammary gland, ovarian, and seminal vesicle tumors were low but significant: 0/106 controls, 3/105 low-dose, and 8/105 high-dose mice presented one of these neoplasms (p = 0.0025). Incidences of females showing any clearly AZT-related neoplasm, in lung, liver, ovary, or mammary gland or histiocytic sarcoma, in the second year, were 12/32 after the low dose and 14/27 after the high dose vs 3/23 controls (p = 0.0045). Also, the sensitivity of neonatal mice was assessed by administration of 25, 50, 100, or 200 mg/kg AZT on postnatal days 1 through 8. The effects at 2 years were similar to those seen after transplacental exposure, with significant increases in lung, liver, and mammary tumors in females. The results confirm that AZT is a moderately effective perinatal carcinogen in mice, targeting several tissue types.

3'-azido-3'-deoxythymidine (AZT) transplacental perfusion kinetics and DNA incorporation in normal human placentas perfused with AZT.

Vertical transmission of the human immunodeficiency virus 1 (HIV-1) is reduced from approximately 25% to approximately 7% as a result of 3'-azido-3'-deoxythymidine (AZT) therapy given during pregnancy; however, the consequences of transplacental AZT exposure to the fetus remain unknown. To address the extent and kinetics of AZT transfer across the human placenta, perfusion studies have been performed with fresh uninfected human placentas perfused with 0.5, 1. 0 and 5.0 mg AZT/ml for 2 h using a dual recirculating single cotyledon perfusion apparatus [T.I. Ala-Kokko, P. Pienimaki, R. Herva, A.I. Hollmen, O. Pelkonen, K. Vähäkangas, Transfer of lidocaine and bupivacaine across the isolated perfused human placenta, Pharmacol. Toxicol. 77 (1995) 142-148]. For two placentas, samples of perfusion effluent were taken every 15 min from the maternal and fetal sides of the apparatus and AZT levels were determined by AZT radioimmunoassay (RIA). At the end of the perfusion, AZT-DNA incorporation into placental DNA was determined by AZT-RIA. The concentration of AZT in the fetal perfusate increased with time, along with a concomitant slow decrease in the concentration of AZT in the maternal perfusates. For three different placentas, at 2 h after the start of perfusion, AZT-DNA incorporation values (molecules of AZT/10(6) nucleotides) were 11.8 for the 0.5 mg AZT/ml perfusate, 13.7 for the 1.0 mg AZT/ml perfusion, and 42.0 for the 5 mg AZT/ml perfusion. An additional placenta perfused with 1 mg AZT/ml did not have detectable values of AZT incorporated into DNA (data not shown). The data show that AZT crosses the human placenta and becomes rapidly incorporated into DNA of placental tissue in a dose-dependent fashion, suggesting that even short exposures to this drug might induce fetal genotoxicity and might also inhibit maternal-fetal viral transmission.

Genotoxicity of 3'-azido-3'-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.

Incorporation of zidovudine into leukocyte DNA from HIV-1-positive adults and pregnant women, and cord blood from infants exposed in utero.

OBJECTIVE: The nucleoside analog 3'-azido-3'-deoxythymidine (ZDV) has widespread clinical use but also is carcinogenic in newborn mice exposed to the drug in utero and becomes incorporated into newborn mouse DNA. This pilot study was designed to determine ZDV incorporation into human blood cell DNA from adults and newborn infants. DESIGN: In this prospective cohort study, peripheral blood mononuclear cells (PBMC) were obtained from 28 non-pregnant adults and 12 pregnant women given ZDV therapy, six non-pregnant adults with no exposure to ZDV, and six non-pregnant adults who last received ZDV > or = 6 months previously. In addition, cord blood leukocytes were obtained from 22 infants of HIV-1-positive, ZDV-exposed women and from 12 infants unexposed to ZDV. There were 11 mother-infant pairs involving HIV-1 -positive women. METHODS: DNA was extracted from PBMC obtained from non-pregnant HIV-1-positive adults taking ZDV, pregnant HIV-1-positive women given ZDV during pregnancy, and from adults not taking ZDV. Cord blood leukocytes were examined from infants exposed to ZDV in utero and from unexposed controls. DNA samples were assayed for ZDV incorporation by anti-ZDV radioimmunoassay (RIA). RESULTS: The majority (76%) of samples from ZDV-exposed individuals, pregnant women (8 of 12), non-pregnant adults (24 of 28), or infants at delivery (15 of 22), had detectable ZDV-DNA levels. The range of positive values for ZDV-treated adults and infants was 25-544 and 22-452 molecules ZDV/10(6) nucleotides, respectively. Analysis of 11 mother-infant pairs showed variable ZDV-DNA incorporation in both, with no correlation by pair or by duration of drug treatment during pregnancy. Two of the 24 samples from individuals designated as controls were positive by anti-ZDV RIA. The 20-fold range for ZDV-DNA values in both adults and infants suggested large interindividual differences in ZDV phosphorylation. CONCLUSIONS: Incorporation of ZDV into DNA was detected in most of the samples from ZDV-exposed adults and infants. Therefore, the biologic significance of ZDV-DNA damage and potential subsequent events, such as mutagenicity, should be

Incorporation of 3'-azido-3'-deoxythymidine (AZT) into fetal DNA and fetal tissue distribution of drug after infusion of pregnant late-term rhesus macaques with a human-equivalent AZT dose.

In the United States, the nucleoside analogue drug 3'-azido-3'deoxythymidine (AZT; also called zidovudine or ZDV) is given to most pregnant women who produce a positive test result for HIV-1. To investigate transplacental distribution and genotoxicity of AZT, near-term pregnant rhesus (Macaca mulatta) monkeys and their fetuses were studied. Four pregnant monkeys were continuously infused with 8 mg AZT/kg body weight for the 4 hours just prior to hysterotomy at term. This short-term AZT exposure resulted in AZT incorporation into DNA of fetal liver, lung, heart, skeletal muscle, brain, testis, and placenta, which varied between 29 and 1944 molecules of AZT/10(6) nucleotides. In contrast, values for AZT and combined metabolites, determined by radioactivity, varied between 0.94 and 5.20 microg AZT equivalents/g tissue. A fifth animal, (H076), was infused with 17.3 mg AZT/kg body weight for approximately 3 hours, followed by 1 hour without drug before hysterotomy. Similar to the 4 other monkeys, variable levels of AZT (16-147 molecules of AZT/10(6) nucleotides) were incorporated into organ DNA of H076, whereas organ tissues contained less-variable levels of AZT and metabolites (0.86-2.05 microg AZT equivalents/g tissue). For H076, at hysterotomy 1 hour after discontinuation of drug, values for AZT and the 3'-azido-3'-deoxythymidine-beta-D-glucuronide (AZTG) in fetal blood and amniotic fluid were twofold and threefold higher than those in maternal blood. Most AZT pharmacokinetic parameters in the fifth monkey were similar to those previously reported for the first 4 monkeys and those observed in a similar study of pregnant women. These data show that a short-term AZT infusion in pregnant rhesus monkeys, which have similar AZT pharmacokinetics to those present in a pregnant human, results in incorporation of drug into the DNA of placenta and most fetal organs. Data imply that the human fetus may also be subject to incorporation of AZT into DNA even after short-term AZT infusion to the mother just before delivery.

Skin tumorigenesis and Ki-ras and Ha-ras mutations in tumors from adult mice exposed in utero to 3'-azido-2',3'-dideoxythymidine.

This study was designed to evaluate the potential initiating effects of transplacental 3'-azido-2',3'-dideoxythymine (AZT) and the role of ras mutational activation in skin tumors induced in a two-stage mouse skin model. In addition, mouse liver and lung tumors from a transplacental AZT tumorigenicity study reported elsewhere (Olivero et al., J Natl Cancer Inst 89:1602-1608, 1997) were examined for evidence of ras activation. For both tumor studies, pregnant CD-1 mice were given either vehicle or 25 mg of AZT daily on days 12-18 of gestation. In the 1997 study, the offspring were given no further exposure and were killed at 1 yr of age. For the skin tumor study, all mice received twice-weekly topical 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment from weeks 5-35; half of the mice had been exposed to AZT in utero. At weeks 16-18, 30, 31, and 34-41, the skin tumor incidences in mice given AZT and TPA were significantly higher than in mice given TPA alone (P < or = 0.05). At week 41, the average numbers of tumors per mouse were 1.44+/-0.36 (mean +/- standard error of the mean) and 0.57+/-0.13 for mice given AZT plus TPA and TPA alone, respectively (P = 0.006). Mutagenesis in ras exons I and II was determined by polymerase chain reaction (PCR) and dye-terminator cycling sequencing of PCR products. Ha-ras exon I codons 12 and 13 were mutated in 11 of 19 tumors (58%) from mice given AZT and TPA and in one of 15 tumors (7%) from mice given TPA alone (P= 0.004). The only mutation in Ha-ras codon 12 (four in four tumors examined) was a G-->A transition in the second base, and the major mutation in codon 13 (six in seven tumors examined) was a G-->T transversion in the second base. In skin tumors, AZT exposure did not increase the number of Ha-ras codon 61 mutations, and no Ki-ras mutations were observed. Analysis of ras mutations in liver and lung tumors from mice exposed to AZT in utero (Olivero et al., J Natl Cancer Inst 89:16021608, 1997) with no TPA promotion showed no significant AZT-related increases.

Irreversible telomere shortening by 3'-azido-2',3'-dideoxythymidine (AZT) treatment.

Telomeres shorten by 30 to 50 bp with each cell division. Germ line, tumor and stem cells overcome progressive shortening by elongating their telomeres with telomerase. Previously we demonstrated that 3'-azido-2',3'-dideoxythymidine (AZT), incorporates into telomeric DNA. To determine if telomeric AZT incorporation was a telomerase mediated phenomenon, we subjected tumor cells to long-term AZT exposure. Here we report the shortening of the telomeric sequences of HeLa cells cultured with 800 microM AZT for 15 passages. Southern blots of HeLa DNA cultured with AZT and digested with SAU 3AI, Alu I, and Rsa I revealed a progressive shortening of the telomeric repeats when probed with a human biotinylated telomeric probe. The shortened telomeric repeats did not elongate after culturing without AZT for an additional 25 passages. No evidence of senescence could be detected.

Transplacental effects of 3'-azido-2',3'-dideoxythymidine (AZT): tumorigenicity in mice and genotoxicity in mice and monkeys.

BACKGROUND: When given during pregnancy, the drug 3'-azido-2',3'-dideoxythymidine (AZT) substantially reduces maternal-fetal transmission of human immunodeficiency virus type 1 (HIV-1). However, AZT has been shown to be carcinogenic in adult mice after lifetime oral administration. In this study, we assessed the transplacental tumorigenic and genotoxic effects of AZT in the offspring of CD-1 mice and Erythrocebus patas monkeys given AZT orally during pregnancy. METHODS: Pregnant mice were given daily doses of either 12.5 or 25.0 mg AZT on days 12 through 18 of gestation (last 37% of gestation period). Pregnant monkeys were given a daily dose of 10.0 mg AZT 5 days a week for the last 9.5-10 weeks of gestation (final 41%-43% of gestation period). AZT incorporation into nuclear and mitochondrial DNA and the length of chromosomal end (telomere) DNA were examined in multiple tissues of newborn mice and fetal monkeys. Additional mice were followed from birth and received no further treatment until subjected to necropsy and complete pathologic examination at 1 year of age. An anti-AZT radioimmunoassay was used to monitor AZT incorporation into DNA. RESULTS: At 1 year of age, the offspring of AZT-treated mice exhibited statistically significant, dose-dependent increases in tumor incidence and tumor multiplicity in the lungs, liver, and female reproductive organs. AZT incorporation into nuclear and mitochondrial DNA was detected in multiple organs of transplacentally exposed mice and monkeys. Shorter chromosomal telomeres were detected in liver and brain tissues from most AZT-exposed newborn mice but not in tissues from fetal monkeys. CONCLUSIONS: AZT is genotoxic in fetal mice and monkeys and is a moderately strong transplacental carcinogen in mice examined at 1 year of age. Careful long-term follow-up of AZT-exposed children would seem to be appropriate.

Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA.

Levels of DNA adducts in Chinese hamster ovary (CHO) cells exposed to cis-diamminedichloroplatinum(II) (cisplatin) for 24 h, have been shown to be 4- to 6-fold higher in mitochondrial (mt) DNA as compared to nuclear (n) DNA (Olivero et al., Mutation Res., 346 (1995) 221). The aim of the present study was to understand if the preferential cisplatin binding in mtDNA is partially caused by lack of adduct removal in the mitochondria. Chinese hamster ovary cells were exposed for 6 h to 50 microM cisplatin, followed by incubation for 24 and 48 h in cisplatin-free medium. At the 30-h time point (6 h with cisplatin, 24 h without cisplatin), half of the cells from each plate were harvested and the remainder were cultured and harvested at 54 h (6 h with cisplatin, 48 h without cisplatin). The 30- and 54-h time points are called 'T30' and 'T54', respectively. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An initial higher level of cisplatin-DNA adducts was observed in mtDNA when compared to nDNA, at T30. In addition, a lack of removal of adducts in mtDNA was demonstrated in cells at T54. Dilution of DNA adducts by DNA replication was documented in pulse-chase experiments that employed [3H]thymidine incorporation. Adduct removal by repair-related mechanisms was considered to comprise the difference between total DNA adduct removal and adduct removal related to DNA replication. The final results demonstrated that both, higher initial binding and lack of removal of cisplatin-DNA adducts appear to contribute to the preferential cisplatin-mtDNA binding observed in CHO cells.