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Primary cilia arise from the centrosomes of quiescent or post-mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) cells. Since cilia are derived from centrosomes, and aneugens can induce centrosomal amplification, the production of multiple cilia arising from multiple centrosomes may reveal the aneugenic nature of the agents. Cells were exposed to AZT to induce centrosomal amplification, cultured without serum to allow the centrioles to develop cilia, and immunostained to visualize cilia and centrosomes. Nuclear DNA was stained with DAPI. Preliminary observations suggest that cells with multiple centrosomes are able to generate extra cilia., (Copyright © 2015 John Wiley & Sons, Inc.)

Background: The objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases., Methods: We performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens., Results: Autoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative., Conclusions: The distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women. Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.

The nucleoside reverse transcriptase inhibitor zidovudine (AZT) induces genotoxic damage that includes centrosomal amplification (CA > 2 centrosomes/cell) and micronucleus (MN) formation. Here we explored these end points in mice deficient in DNA repair and tumor suppressor function to evaluate their effect on AZT-induced DNA damage. We used mesenchymal-derived fibroblasts cultured from C57BL/6J mice that were null and wild type (WT) for Xpa, and WT, haploinsufficient and null for p53 (6 different genotypes). Dose-responses for CA formation, in cells exposed to 0, 10, and 100 μM AZT for 24 hr, were observed in all genotypes except the Xpa((+/+)) p53((+/-)) cells, which had very low levels of CA, and the Xpa((-/-)) p53((-/-)) cells, which had very high levels of CA. For CA there was a significant three-way interaction between Xpa, p53, and AZT concentration, and Xpa((-/-)) cells had significantly higher levels of CA than Xpa((+/+)) cells, only for p53((+/-)) cells. In contrast, the MN and MN + chromosomes (MN + C) data showed a lack of AZT dose response. The Xpa((-/-)) cells, with p53((+/+)) or ((+/-)) genotypes, had levels of MN and MN + C higher than the corresponding Xpa((+/+)) cells. The data show that CA is a major event induced by exposure to AZT in these cells, and that there is a complicated relationship between AZT and CA formation with respect to gene dosage of Xpa and p53. The loss of both genes resulted in high levels of damage, and p53 haploinsufficicency strongly protected Xpa((+/+)) cells from AZT-induced CA damage., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

The cytokinesis-block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT-3 human lymphoblastoid cells exposed to 10 μM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR-1065 and Tempol, to decrease AZT-induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR-1065 would protect against AZT-induced genotoxicity. MOLT-3 cells were exposed to 0 or 10 µM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 µM WR-1065 and/or 0 or 200 µM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT-3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR-1065 protected against AZT-induced MN formation (P < 0.003 for both), and WR-1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR-1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR-1065 and Tempol protected MOLT-3 cells against specific types of AZT-induced DNA damage., (© 2014 Wiley Periodicals, Inc.)

Background: Erythrocebus patas (patas) monkeys were used to model antiretroviral (ARV) drug in human immunodeficiency virus type 1-infected pregnant women., Methods: Pregnant patas dams were given human-equivalent doses of ARVs daily during 50% of gestation. Mesenchymal cells, cultured from bone marrow of patas offspring obtained at birth and at 1 and 3 years of age, were examined for genotoxicity, including centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes., Results: Compared with controls, statistically significant increases (P < .05) in centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes were found in mesenchymal cells from most groups of offspring at the 3 time points., Conclusions: Transplacental nucleoside reverse-transcriptase inhibitor exposures induced fetal genotoxicity that was persistent for 3 years.

We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(-/-)p53(+/-) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N (2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(-/-)p53(+/-) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(-/-)p53(+/-) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(-/-)p53(+/-) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP-DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(-/-)p53(+/-) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH-DNA adduct levels consistently in human organs.

Three classes of DNA damage were assessed in human placentas collected (2000-2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by (32)P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49-312 PAH-DNA adducts/10(8) nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7-8.6 stable/bulky DNA adducts/10(8) nucleotides and 0.6-47.2 AB sites/10(5) nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi., (Copyright © 2010 Wiley-Liss, Inc.)

This collection of articles was inspired by the long-standing relationship between the Environmental Mutagen Society and Latin American scientists, and by the program for the 39th Environmental Mutagen Society meeting in Puerto Rico in 2008, which included a symposium featuring "South of the border" scientists. This collection, compiled by Graciela Spivak and Ofelia Olivero, both originally from Argentina, highlights scientists who work in or were trained in Latin American countries and in Puerto Rico in a variety of scientific specialties related to DNA repair and cancer susceptibility, genomic organization and stability, genetic diversity, and environmental contaminants.

The antiretroviral efficacy of 3'-azido-3'-deoxythymidine (AZT) is dependent upon intracellular mono-, di-, and triphosphorylation and incorporation into DNA in place of thymidine. Thymidine kinase 1 (TK-1) catalyzes the first step of this pathway. MOLT-3, human lymphoblastoid cells, were exposed to AZT continuously for 14 passages (P(1)-P(14)) and cultured for an additional 14 passages (P(15)-P(28)) without AZT. Progressive and irreversible depletion of the enzymatically active form of the TK-1 24-kDa monomer with loss of active protein was demonstrated during P(1)-P(5) of AZT exposure. From P(15) to P(28), both the 24- and the 48-kDa forms of TK-1 were undetectable and a tetrameric 96-kDa form was present. AZT-DNA incorporation was observed with values of 150, 133, and 108 molecules of AZT/10(6) nucleotides at the 10 microM plasma-equivalent AZT dose at P(1), P(5), and P(14), respectively. An exposure-related increase in the frequency of micronuclei (MN) was observed in cells exposed to either 10 or 800 microM AZT during P(1)-P(14). Analysis of the cell cycle profile revealed an accumulation of S-phase cells and a decrease in G(1)-phase cells during exposure to 800 microM AZT for 14 passages. When MOLT-3 cells were grown in AZT-free media (P(15)-P(29)), there was a reduction in AZT-DNA incorporation and MN formation; however, TK-1 depletion and the persistence of S-phase delay were unchanged. These data suggest that in addition to known mutagenic mechanisms, cells may become resistant to AZT partially through inactivation of TK-1 and through modulation of cell cycle components.

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., (Copyright 2010 S. Karger AG, Basel.)

Background: WR1065 is the free-thiol metabolite of the cytoprotective aminothiol amifostine, which is used clinically at very high doses to protect patients against toxicity induced by radiation and chemotherapy. In an earlier study we briefly reported that the aminothiol WR1065 also inhibits HIV-1 replication in phytohemagglutinin (PHA)-stimulated human T-cell blasts (TCBs) infected in culture for 2 hr before WR1065 exposure. In this study we expanded the original observations to define the dose-response curve for that inhibition, and address the question of additive effects for the combination of WR1065 plus Zidovudine (AZT). Here we also explored the effect of WR1065 on SIV by examining TCBs taken from macaques with well-established infections several months with SIV., Results: TCBs from healthy human donors were infected for 2 hr with HIV-1, and viral replication (p24) was measured after 72 hr of incubation with or without WR1065, AZT, or both drugs. HIV-1 replication, in HIV-1-infected human TCBs, was inhibited by 50% at 13 microM WR1065, a dose at which 80% of the cells were viable. Cell cycle parameters were the same or equivalent at 0, 9.5 and 18.7 microM WR1065, showing no drug-related toxicity. Combination of AZT with WR1065 showed that AZT retained antiretroviral potency in the presence of WR1065. Cultured CD8+ T cell-depleted PHA-stimulated TCBs from Macaca mulatta monkeys chronically infected with SIV were incubated 17 days with WR1065, and viral replication (p27) and cell viability were determined. Complete inhibition (100%) of SIV replication (p27) was observed when TCBs from 3 monkeys were incubated for 17 days with 18.7 microM WR1065. A lower dose, 9.5 microM WR1065, completely inhibited SIV replication in 2 of the 3 monkeys, but cells from the third macaque, with the highest viral titer, only responded at the high WR1065 dose., Conclusion: The study demonstrates that WR1065 and the parent drug amifostine, the FDA-approved drug Ethyol, have antiretroviral activity. WR1065 was active against both an acute infection of HIV-1 and a chronic infection of SIV. The data suggest that the non-toxic drug amifostine may be a useful antiretroviral agent given either alone or in combination with other drugs as adjuvant therapy.

In cultured cells, exposure to the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induces genomic instability, cell cycle arrest, micronuclei, sister chromatid exchanges, and shortened telomeres. In previous studies, we demonstrated AZT-induced centrosome amplification (>2 centrosomes/cell). Here, we investigate centrosome amplification in cells exposed to other commonly used NRTIs. Experiments were performed using Chinese Hamster ovary (CHO) cells, and two normal human mammary epithelial cell (NHMEC) strains: M99005 and M98040, which are high and low incorporators of AZT into DNA, respectively. Cells were exposed for 24 hr to lamivudine (3TC), stavudine (d4T), didanosine (ddI), and thymidine, and stained with anti-pericentrin antibody. Dose response curves were performed to determine cytotoxicity and a lower concentration at near plasma levels and a 10 fold higher concentration were chosen for the experiments. In CHO cells, there was a concentration-dependent, significant (P < 0.05) increase in centrosome amplification for each of the NRTIs. In NHMEC strain M99005, an NRTI-induced increase (P < 0.05) in centrosome amplification was observed for the high concentrations of each NRTI and the low doses of 3TC and ddI. In NHMEC strain M98040, the high doses of ddI and d4T showed significant increases in centrosome amplification. Functional viability of amplified centrosomes was assessed by arresting microtubule nucleation with nocodazole. In cells with more than two centrosomes, the ability to recover microtubule nucleation was similar to that of unexposed cells. We conclude that centrosome amplification is a consequence of exposure to NRTIs and that cells with centrosome amplification are able to accomplish cell division., ((c) 2009 Wiley-Liss, Inc.)

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation., (Copyright 2009 Wiley-Liss, Inc.)

The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese hamster ovary (CHO) and normal human mammary epithelial cells (NHMECs) exposed to the antiretroviral drug zidovudine (3'-azido-3'-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC) strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (high incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound.

Use of tamoxifen is associated with a 50% reduction in breast cancer incidence and an increase in endometrial cancer incidence. Here, we documented tamoxifen-induced gene expression changes in cultured normal human mammary epithelial cells (strains 5, 16, and 40), established from tissue taken at reduction mammoplasty from three individuals. Cells exposed to 0, 10, or 50 micromol/L of tamoxifen for 48 hours were evaluated for (E)-alpha-(deoxyguanosine-N(2)-yl)-tamoxifen (dG-N(2)-TAM) adduct formation using TAM-DNA (DNA modified with dG-N(2)-TAM) chemiluminescence immunoassay, gene expression changes using National Cancer Institute DNA-oligonucleotide microarray, and real-time PCR. At 48 hours, cells exposed to 10 and 50 micromol/L of tamoxifen were 85.6% and 48.4% viable, respectively, and there were no measurable dG-N(2)-TAM adducts. For microarrays, cells were exposed to 10 micromol/L of tamoxifen and genes with expression changes of >3-fold were as follows: 13 genes up-regulated and 1 down-regulated for strain 16; 17 genes up-regulated for strain 5, and 11 genes up-regulated for strain 40. Interferon-inducible genes (IFITM1, IFIT1, MXI, and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all three strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. Real-time PCR revealed the up-regulation of IFNA1 and confirmed the tamoxifen-induced up-regulation of the five other genes identified by microarray, with the exception of GIP3 and MX1, which were not up-regulated in strain 40. Induction of IFN-related genes in the three normal human mammary epithelial cell strains suggests that, in addition to hormonal effects, tamoxifen exposure may enhance immune response in normal breast tissue.

A mainstay of the antiretroviral drugs used for therapy of HIV-1, zidovudine (AZT) is genotoxic and becomes incorporated into DNA. Here we explored host inter-individual variability in AZT-DNA incorporation, by AZT radioimmunoassay (RIA), using 19 different strains of normal human mammary epithelial cells (NHMECs) exposed for 24 h to 200 microM AZT. Twelve of the 19 NHMEC strains showed detectable AZT-DNA incorporation levels (16 to 259 molecules of AZT/10(6) nucleotides), while 7 NHMEC strains did not show detectable AZT-DNA incorporation. In order to explore the basis for this variability, we compared the 2 NHMEC strains that showed the highest levels of AZT-DNA incorporation (H1 and H2) with 2 strains showing no detectable AZT-DNA incorporation (L1 and L2). All 4 strains had similar (> or =80%) cell survival, low levels of accumulation of cells in S-phase, and no relevant differences in response to the direct-acting mutagen bleomycin (BLM). Finally, when levels of thymidine kinase 1 (TK1), the first enzyme in the pathway for incorporation of AZT into DNA, were determined by Western blot analysis in all 19 NHMEC strains at 24 h of AZT exposure, higher TK1 protein levels were found in the 12 strains showing AZT-DNA incorporation, compared to the 7 showing no incorporation (p=0.0005, Mann-Whitney test). Furthermore, strains L1 and L2, which did not show AZT-DNA incorporation at 24 h, did have measurable incorporation by 48 and 72 h. These data suggest that variability in AZT-DNA incorporation may be modulated by inter-individual differences in the rate of induction of TK1 in response to AZT exposure.

The current incidence of human immunodeficiency virus (HIV-1)/AIDS affects around 7000 pregnant women in the United States. When given during pregnancy, the nucleoside analog 3'-azido-3'-deoxythymidine (AZT) significantly reduces maternal-fetal transmission. It has been previously shown that AZT is incorporated into DNA, where it causes mutations in the HPRT and TK genes. It also changes cell cycle gene expression, and induces S-phase arrest, micronuclei, chromosomal aberrations, sister chromatid exchanges, telomeric attrition, and other genotoxic effects in cultured cells. A predicted consequence of these events is genomic instability that together, with clastogenicity may contribute to the carcinogenic potency of AZT. Various aspects of genotoxicity are explored in this contribution seeking to understand the multiple effects of this antiretroviral agent in animal models and humans. This mini-review describes some of the experimental models used to elucidate the genotoxicity induced by antiretroviral therapy during human pregnancy. The use of diverse methods to detect biomarkers of exposure, such as an AZT-specific radioimmunoassay, micronuclei bearing intact chromosomes, and telomeric DNA attrition highlight the role of in vitro models to elucidate exposure and risk. The relevance of the in vitro models is followed by the introduction of the role of the nucleoside analogs in transplacental carcinogenesis along with the description of a transplacental perfusion model and a transplacental carcinogenesis rodent model. In a more direct clinical application the use of AZT-DNA incorporation as a biomarker of exposure, in experiments conducted in vivo in Erythrocebus patas monkeys and in humans, addresses the possibility of elucidation of potential cancer risk in those infants exposed in utero. Two relevant aspects of this contribution are the potential application of some of the models described in this mini-review, as diagnostic tools in antiretroviral-exposed populations, and the use of these models to understand the nature of the genotoxicities and minimize the undesirable side effects of the antiretroviral therapy.

Among women infected with carcinogenic human papillomavirus (HPV), there is a two- to five-fold increased risk of cervical precancer and cancer in women who smoke compared to those who do not smoke. Because tobacco smoke contains carcinogenic polycyclic aromatic hydrocarbons (PAHs), it was of interest to examine human cervical tissue for PAH-DNA adduct formation. Here, we measured PAH-DNA adduct formation in cervical biopsies collected in follow-up among women who tested positive for carcinogenic HPV at baseline. A semi-quantitative immunohistochemistry (IHC) method using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was used to measure nuclear PAH-DNA adduct formation. Cultured human cervical keratinocytes exposed to 0, 0.153, or 0.331microM BPDE showed dose-dependent increases in r7,t8,t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adducts. For BPdG adduct analysis, paraffin-embedded keratinocytes were stained by IHC with analysis of nuclear color intensity by Automated Cellular Imaging System (ACIS) and, in parallel cultures, extracted DNA was assayed by quantitative BPDE-DNA chemiluminescence immunoassay (CIA). For paraffin-embedded samples from carcinogenic HPV-infected women, normal-appearing cervical squamous epithelium suitable for scoring was found in samples from 75 of the 114 individuals, including 29 cases of cervical precancer or cancer and 46 controls. With a lower limit of detection of 20 adducts/10(8) nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/10(8) nucleotides, with a median of 75/10(8) nucleotides. PAH-DNA adduct values above 150/10(8) nucleotides were found in eight samples, and in three samples adducts were non-detectable. There was no correlation between PAH-DNA adduct formation and either smoking or case status. Therefore, PAH-DNA adduct formation as measured by this methodology did not appear related to the increased risk of cervical precancer and cancer among carcinogenic HPV-infected smokers.

Several systemic and cellular markers of 3'-azido-3'-dideoxythymidine (AZT) metabolism and AZT incorporation into nuclear DNA were measured in cord blood from uninfected infants born to HIV-1-infected mothers receiving prepartum therapies based on AZT or AZT in combination with 2',3'-dideoxy-3'-thiacytidine (3TC). In addition, the relationships among these pharmacological end points, levels of AZT-DNA incorporation, and the previously reported mutagenic responses in these infants were evaluated. AZT- and 3TC-specific radioimmunoassays (RIAs), or HPLC coupled with AZT-RIA, were used to measure plasma levels of AZT and the AZT-glucuronide, and cellular levels of AZT, phosphorylated AZT, and DNA incorporation of AZT or 3TC in cord blood mononuclear cells from treated infants compared with unexposed controls born to HIV-uninfected mothers. Fewer infants had detectable AZT-DNA incorporation levels in the group exposed to AZT (71%; n = 7) compared with those receiving AZT-3TC (100%; n = 21), and the mean AZT-DNA incorporation for AZT-exposed infants (14.6 +/- 6.3 AZT/10(6) nucleotides) was significantly lower than that in AZT-3TC exposed infants (51.6 +/- 10.2 AZT/10(6) nucleotides; P = 0.028). Low levels of 3TC-DNA incorporation found in a few AZT-3TC-exposed newborns correlated with AZT-DNA incorporation values in the same samples. Among the metabolites studied, there were positive correlations between levels of AZT-diphosphate and AZT-triphosphate, and AZT-triphosphate and AZT-DNA incorporation, in nucleoside analog-exposed infants. Levels of AZT-DNA incorporation, however, did not correlate well with the reported frequencies of somatic mutations in the same population of nucleoside analog-treated children. While these data support the continued use of AZT-based therapies during pregnancy, infants receiving prepartum AZT should be monitored long-term for adverse health effects., ((c) 2006 Wiley-Liss, Inc.)

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., ((c) 2006 Wiley-Liss, Inc.)

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., ((c) 2006 Wiley-Liss, Inc.)

Experiments were performed to investigate the impact of zidovudine (AZT), lamivudine (3TC), and abacavir (ABC) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using cell cloning assays for assessing the effects of individual drugs/drug combinations in (1) TK6 human lymphoblastoid cells exposed in vitro and (2) splenic lymphocytes from male CD-1 mice exposed transplacentally on days 12-18 of gestation. In TK6 cells, dose-related increases in HPRT and TK mutant frequencies were found following 3 days of exposure to AZT or 3TC alone (33, 100, or 300 microM), or to equimolar amounts of AZT-3TC. Compared with single drug exposures, AZT-3TC coexposures generally yielded enhanced elevations in HPRT and TK mutant frequencies. Mutagenicity experiments with ABC alone, or in combination with AZT-3TC, were complicated by the extreme cytotoxicity of ABC. Exposure of cells either to relatively high levels of AZT-3TC short-term (100 microM, 3 days), or to peak plasma-equivalent levels of AZT-3TC for an extended period (10 microM, 30 days), resulted in similar drug-induced mutagenic responses. Among sets of mice necropsied on days 13, 15, or 21 postpartum, Hprt mutant frequencies in T-cells were significantly elevated in the AZT-only (200 mg/kg bw/day) and AZT-3TC (200 mg AZT + 100 mg 3TC/kg bw/day) groups at 13 days of age. These results suggest that the mutagenicity by these nucleoside analogs is driven by cumulative dose, and raises the question of whether AZT-3TC has greater mutagenic effects than AZT alone in perinatally exposed children., ((c) 2006 Wiley-Liss, Inc.)

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., ((c) 2006 Wiley-Liss, Inc.)

In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.

Microarrays are used to study gene expression in a variety of biological systems. A number of different platforms have been developed, but few studies exist that have directly compared the performance of one platform with another. The goal of this study was to determine array variation by analyzing the same RNA samples with three different array platforms. Using gene expression responses to benzo[a]pyrene exposure in normal human mammary epithelial cells (NHMECs), we compared the results of gene expression profiling using three microarray platforms: photolithographic oligonucleotide arrays (Affymetrix), spotted oligonucleotide arrays (Amersham), and spotted cDNA arrays (NCI). While most previous reports comparing microarrays have analyzed pre-existing data from different platforms, this comparison study used the same sample assayed on all three platforms, allowing for analysis of variation from each array platform. In general, poor correlation was found with corresponding measurements from each platform. Each platform yielded different gene expression profiles, suggesting that while microarray analysis is a useful discovery tool, further validation is needed to extrapolate results for broad use of the data. Also, microarray variability needs to be taken into consideration, not only in the data analysis but also in specific probe selection for each array type.

Antiretroviral therapy for the human immunodeficiency virus-1 (HIV-1) typically includes two nucleoside reverse transcriptase inhibitors (NRTIs). 3'-Azido-3'-deoxythymidine (AZT, Zidovudine) plus 2'-deoxy-3'-thiacytidine (3TC, Lamivudine) is a combination that is used frequently. The NRTIs are mutagenic nucleoside analogs that become incorporated into DNA and terminate replication. We therefore hypothesized that exposure to this class of drug may alter cell cycle parameters. We used flow cytometry to examine the cell cycle in human epithelioid carcinoma (HeLa) cells exposed to AZT and 3TC alone, as well as a series of AZT/3TC dose combinations: (A) 125.0 microM AZT/12.5 microM 3TC; (B) 250.0 microM AZT/25.0 microM 3TC; and (C) 500 microM AZT/50 microM 3TC. At 24 h, at all doses, there was a good cell viability (>/=68%), and incorporation of AZT into nuclear DNA. Using flow cytometry, a dose-related increase in the percentage of cells in S phase, from 9.5% with no drug, to 36.0% with dose C, was observed in cells exposed for 24 h (P = 0.001, ANOVA). A concomitant decrease in the percentage of cells in G(1) phase, from 82.6% with no drug to 58.5% with dose C, was observed in cells exposed for 24 h (P = 0.017, ANOVA). A similar S phase arrest was seen in cells exposed to 125, 250 and 500 microM AZT alone, but there was no S phase alteration with 50 microM 3TC alone, suggesting that AZT is responsible for the accumulation of cells in S phase. To elucidate the accumulation of cells in S phase and explore the cell cycle gene expression changes induced by AZT and 3TC, we used c-DNA microarray, Cell Cycle Super Array and real-time PCR. There was a strong upregulation of the DNA damage-inducible transcript 3 (DDIT3 or GADD153) in NRTI-exposed cells. In addition, AZT induced an upregulation of cyclin D1 accompanied by a downregulation of the cyclin D1-associated inhibitors P18 and P57, and the G(1)-S check point gene P21, the net effect of which would be to foster a cell progression into S phase. Cyclin A2 was down-regulated in cells exposed to AZT, suggesting a block in S-G(2)-M progression that would also be consistent with the accumulation of cells in S phase. Overall, the study demonstrates that AZT, but not 3TC, causes an arrest of cells in S phase with a consistent alteration in the expression of several cell cycle genes.

The current worldwide spread of the human immunodeficiency virus-1 (HIV-1) to the heterosexual population has resulted in approximately 800,000 children born yearly to HIV-1-infected mothers. In the absence of anti-retroviral intervention, about 25% of the approximately 7,000 children born yearly to HIV-1-infected women in the United States are HIV-1 infected. Administration of zidovudine (AZT) prophylaxis during pregnancy reduces the rate of infant HIV-1 infection to approximately 7%, and further reductions are achieved with the addition of lamivudine (3TC) in the clinical formulation Combivir. Whereas clinically this is a remarkable achievement, AZT and 3TC are DNA replication chain terminators known to induce various types of genotoxicity. Studies in rodents have demonstrated AZT-DNA incorporation, HPRT mutagenesis, telomere shortening, and tumorigenicity in organs of fetal mice exposed transplacentally to AZT. In monkeys, both AZT and 3TC become incorporated into the DNA from multiple fetal organs taken at birth after administration of human-equivalent protocols to pregnant dams during gestation, and telomere shortening has been found in monkey fetuses exposed to both drugs. In human infants, AZT-DNA and 3TC-DNA incorporation as well as HPRT and GPA mutagenesis have been documented in cord blood from infants exposed in utero to Combivir. In infants of mice, monkeys, and humans, levels of AZT-DNA incorporation were remarkably similar, and in newborn mice and humans, mutation frequencies were also very similar. Given the risk-benefit ratio, these highly successful drugs will continue to be used for prevention of vertical viral transmission, however evidence of genotoxicity in mouse and monkey models and in the infants themselves would suggest that exposed children should be followed well past adolescence for early detection of potential cancer hazard.

This study was designed to investigate fetal mitochondrial toxicity in Erythrocebus patas monkeys exposed in utero to zidovudine (AZT) and lamivudine (3TC), and taken at term. Pregnant patas monkeys were given a daily dose of 40 mg AZT (86% of the human daily dose, based on body weight), for the last 10 weeks (50%) of gestation, and a daily dose of 24 mg 3TC (84% of the human daily dose, based on body weight) for the last 4 weeks of gestation. At term, AZT was found to be incorporated into fetal mitochondrial DNA from skeletal muscle, liver, kidney, and placenta. By transmission electron microscopy (EM) drug-exposed fetal cardiac and skeletal muscle cells showed mitochondrial membrane compromise, mitochondrial proliferation, and damaged sarcomeres, while mitochondria in brain cerebrum and cerebellum were morphologically normal. Substantial depletion of oxidative phosphorylation (OXPHOS) Complex I specific activities was observed in heart (87% reduction in mean, p = 0.02) and skeletal muscle (98% reduction in mean, p = 0.002) from drug-exposed fetuses, compared to unexposed fetuses. In addition Complex IV activity was highly depleted (85% reduction in mean, p = 0.004) in skeletal muscle from the drug-exposed fetuses (p = 0.004). Brain cerebrum and cerebellum showed no statistically significant OXPHOS changes with drug exposure. Mitochondrial DNA quantity was substantially depleted (>50%) in heart, skeletal muscle, cerebellum, and cerebrum from drug-exposed fetuses compared to unexposed controls. Overall, the data indicate that significant mitochondrial damage was observed at birth in monkey fetuses exposed in utero to AZT plus 3TC in a human-equivalent dosing protocol.

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.

It has been suggested that ingestion of polycyclic aromatic hydrocarbons (PAHs) may contribute to the high incidence and mortality of esophageal cancer in Linxian, China. To explore this relationship a semiquantitative immunohistochemical staining method was developed for localization of PAH-DNA adducts. Nuclear color intensity (bright field average pink intensity per nucleus for >1000 cells) was measured using the ChromaVision Automated Cellular Imaging System (ACIS). Paraffin-embedded sections of cultured human keratinocytes exposed to increasing concentrations of 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) were incubated with BPDE-DNA antiserum and served as an internal positive control (standard curve). Values for nuclear staining intensity correlated directly with BPDE exposure concentration (r(2) = 0.99) and were reproducible. DNA adduct levels determined by BPDE-DNA chemiluminescence immunoassay in DNA from BPDE-exposed keratinocytes, correlated with BPDE exposure concentrations (r(2) = 0.99), showing that nuclear staining intensity determined by ACIS correlated directly with BPDE-DNA adduct levels determined by chemiluminescence immunoassay. The ACIS methodology was applied to 5 human samples from Linxian, and significantly positive nuclear PAH-DNA adduct staining was observed in this group when compared with esophageal tissue from 4 laboratory-housed monkey controls and 6 samples obtained at autopsy from smokers and nonsmokers in the United States. Nuclear PAH-DNA staining was absent from Linxian samples when serial sections were incubated with normal rabbit serum (negative control) and was significantly reduced on incubation with BPDE-DNA antiserum absorbed previously with the immunogen BPDE-DNA. These results appear to support the hypothesis that high PAH exposure levels may be etiologically associated with the development of esophageal cancer in Linxian.

Antiretroviral nucleoside analogue drugs are a major constituent of highly active antiretroviral therapy (HAART), the most advanced form of treatment for HIV-1 infection. Currently, HAART combinations that include zidovudine (ZDV) and lamivudine (3TC) are highly effective in preventing HIV-1 vertical transmission; most children are born with no evident adverse clinical effects. However, ZDV is a moderately strong transplacental carcinogen in mice, and potential long-term consequences of fetal exposure to most HAART combinations remain unknown. To model human transplacental ZDV and 3TC exposures, experiments were performed in Erythrocebus patas monkeys given human-equivalent drug exposure protocols. Pregnant monkeys were dosed with either no drug (n = 2), 40.0 mg ZDV/d (about 6 mg/kg body weight/d) for the last 50% (10 weeks) of gestation (n = 3), or with the same regimen of ZDV plus 24.0 mg 3TC/d (about 3.6 mg/kg body weight/d) for the last 20% (4 weeks) of gestation (n = 3). Multiple fetal organs were examined at term for DNA incorporation of ZDV and 3TC using two separate radioimmunoassays (RIAs). Values for ZDV-DNA incorporation were similar in fetuses exposed to ZDV alone and those exposed to ZDV plus 3TC. Values for 3TC-DNA in fetal organs were greater than or equal to values for ZDV-DNA, indicating that the total DNA damage sustained by fetuses exposed to both drugs was at least double that observed in fetuses exposed to ZDV alone. Telomere shortening, determined by Southern blot with a telomeric probe, was observed in most organs of the three animals exposed in utero to ZDV plus 3TC. No telomere shortening was evident in the unexposed fetuses, and occasional telomere shortening was found in fetuses exposed to ZDV alone. Overall, these studies demonstrate that monkey fetuses exposed in utero to the combination ZDV plus 3TC sustain a higher level of drug-DNA incorporation and show evidence of more telomere damage than monkey fetuses exposed to ZDV alone.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.