AZT Treatment Research Articles

AZT induces oxidative damage to cardiac mitochondria: Protective effect of vitamins C and E

AZT (zidovudine) is a potent inhibitor of HIV replication and a major antiretroviral drug used for AIDS treatment. A major limitation in the use of AZT is the occurrence of severe side effects. The aim of this work was to test whether AZT causes oxidative damage to heart mitochondria and whether this can be prevented by supranutritional doses of antioxidant vitamins. An experimental animal model was used in which mice were treated with AZT for 35 days (10 mg/kg/day) in drinking water. Animals treated with antioxidant vitamins were fed the same diet as controls but supplemented with vitamins C (ascorbic acid, 10 g/ kg diet) and E (α-dl-tocopherol, 0.6 g/kg diet) for 65 days before sacrifice. This resulted in a daily intake of 1250 mg/kg/day (vitamin C) and 75 mg/kg/day (vitamin E). Cardiac mitochondrial DNA (mtDNA) of mice treated with AZT had over 120% more oxo-dG (8-oxo-7,8-dihydro-2′-deoxyguanosine, which is a biomarker of oxidative damage to DNA) in their mitochondrial DNA than untreated controls. AZT treatment also caused an increase in mitochondrial lipid peroxidation and an oxidation of mitochondrial glutathione. Dietary supplementation with supranutritional doses of the antioxidant vitamins C and E protected against these signs of mitochondrial oxidative stress. The oxidative effects of AZT are probably due to an increase in production of reactive oxygen species by mitochondria of AZT-treated animals, raising the possibility that oxidative stress may play an important role in the cardiotoxicity of AZT.

307. A Mechanism for AZT Mediated Enhancement of Transgene Expression

Recombinant adeno-associated viral (rAAV) vectors have been used to treat hemophilia B by efficient transfer of F.IX into liver and muscle in animals; human trials are currently underway. In a recent dose-escalation study investigating muscle directed F.IX gene transfer using rAAV vectors, an HIV-positive subject who received the lowest vector dose showed highest F.IX expression levels. Coadministration of Zidovudine (3'-Azido-3'-deoxythymidine or AZT) during the trial was suggested as an explanation for this observation (Manno et al, Blood 101:8, 2003). AZT is a nucleoside analog that inhibits HIV reverse transcriptase activity. It has also been implicated in inhibiting DNA replication, in generation of oxygen radicals and in induction of transcription factors. We hypothesized a role for the AZT induced nuclear transcription factor, NF-kappaB, in enhancing gene expression by binding to the four consensus NF-kappaB binding sites in the CMV promoter of the vector. Previously, we have shown an AZT dependent increase (30-fold) in expression of a CMV promoter-driven transgene following transduction of HepG2 cells with an AAV vector. Similar experiments using a vector with an EF1alpha promoter showed no change in levels of transgene expression in response to AZT, indicating a CMV promoter-specific response. Western analysis of total cellular extracts from transduced cells cultured for up to 72h in the presence of different levels of AZT showed an increase in NF-kappaB (p65) levels with increasing AZT. NF-kappaB consensus sequence binding assays using nuclear extracts from AZT treated (0.4, 4 and 40mM) cells showed 1.5, 4.1 and 4.3-fold increase, respectively, in NF-kappaB levels over untreated samples. Reporter assays were performed using plasmids with a luciferase gene driven by either a TK (thymidine kinase) minimal promoter or an NF-kB4-TK promoter. Our results show increasing reporter expression (8-fold in 40mM AZT treatment) with increasing concentrations of AZT with NF-kB4-TK promoter, but not with the TK-minimal promoter. We further confirmed our findings by silencing the ATM gene, an activator of NF-kappaB, using siRNA. ATM silenced cells produced significantly lower levels of active NF-kappaB in nuclear extracts of cells treated with AZT. Moreover, the activity of the NF-kB4-TK luciferase promoter decreased 7-fold, indicating a specific role for ATM in CMV driven gene expression via NF-kappaB activation pathway. Our findings thus provide a molecular explanation for enhanced CMV-promoter driven gene expression in a clinical trial subject treated with Zidovudine and provide a novel avenue towards exploring the use of host transcription factors to significantly enhance gene transfer treatment strategies.

Changes in hematologic parameters and efficacy of thymidine analogue-based, highly active antiretroviral therapy: A meta-analysis of six prospective, randomized, comparative studies

Background: Hematologic changes that occur with HIV infection and antiretroviral therapy may impact the quality of life of patients and have been associated with morbidity and mortality. The management of anemia and neutropenia has improved survival in persons with HIV. Objectives: This meta-analysis was performed to assess the changes in hemoglobin (Hb) levels and neutrophil counts that occur during thymidine analogue-based triple therapy and to establish whether different regimens of triple therapy are similar in efficacy based on CD4 + T-cell count and viral load changes. Methods: This was a meta-analysis of the impact of zidovudine (AZT) compared with stavudine (d4T) in triple therapy regimens in HIV patients with regard to hematologic parameters and efficacy markers. The peer-reviewed literature was searched with use of MEDLINE, PubMed, EMBASE, and the Cochrane database of systematic reviews (key terms: HIV, antiretroviral therapy, CD4, viral load, three, triple, and highly active antiretroviral therapy [HAART]). Searches were initially not limited by publication type, study design, date, or language but subsequently were sorted to include only studies performed in treatment-naive adult patients; randomized; and comparative of a regimen that included d4T with a regimen that included AZT; to involve regimens that included a minimum of 3 drugs each; and to include hematologic outcomes data. Outcomes were CD4 +, viral load, and hematologic parameters. Results: This meta-analysis included 6 studies. Treatment efficacy as measured by changes in CD4 + T-cell counts and viral load did not differ significantly between regimens. Hb levels decreased with AZT treatment by a mean (SE) 0.4 (0.05) g/dL and 0.2 (0.06) g/dL at weeks 24 and 48, respectively, but increased with d4T treatment by 0.45 (0.03) g/dL and 0.58 (0.04) g/dL, respectively. All grades of anemia and neutropenia events were consistently more common with AZT-based regimens relative to d4T-based therapy. Conclusions: AZT-based HAART has a greater negative impact on hematologic parameters relative to d4T-based regimens. AZT recipients are more likely than d4T recipients to experience anemia and neutropenia events of any grade, and AZT is associated with a net decrease in Hb level relative to a net increase with d4T. These factors may influence the choice of drug used in the treatment of certain patient populations.

Anti-HIV drugs decrease the expression of matrix metalloproteinases in astrocytes and microglia.

The introduction of potent antiretroviral drugs for the treatment of patients with human immunodeficiency virus (HIV) infection has dramatically reduced the prevalence of HIV-associated neurological disorders. Such diseases can be mediated by proteolytic enzymes, i.e. matrix metalloproteinases (MMPs) and, in particular gelatinases, released from glial cells. The aim of this study was to investigate whether the antiretroviral drugs commonly used for the treatment of HIV-infected patients modulate the activity of MMPs in astrocyte and microglial cultures. Primary cultures of rat astrocyte and microglia were treated with different doses of zidovudine (AZT) or indinavir (IDV) for 20 h and simultaneously activated by exposure to lipopolysaccharide (LPS). Culture supernatants collected from astrocytes and microglia after 24 h incubation were subjected to gelatin zymography and western blot analysis for the assessment of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) protein levels. Total RNA was extracted from glial cells and used for reverse transcriptase-polymerase chain reaction for the assessment of mRNA expression. Our results indicate that both astrocyte and microglial cells constitutively express MMP-2 mRNA and protein. LPS treatment increased MMP-2 mRNA and protein expression in astrocytes, but not in microglial cells. The treatment with both AZT and IDV dose-dependently inhibited the expression of MMP-2 in astrocytes, whereas it had no effect on microglial cells. The expression of MMP-9 in both astrocytes and microglia was induced by LPS treatment and was dose-dependently inhibited by AZT and IDV treatment in LPS-stimulated astrocytes and microglia. These results raise the possibility that AZT and IDV interfere directly with MMP production in glial cells and independently from their antiviral activity, thus suggesting the possible therapeutical use in neurological diseases associated with MMPs involvement.

AZT inhibits Visna/maedi virus-induced apoptosis.

Visna/maedi virus (VMV) causes severe encephalitis and a progressive demyelinating disease in sheep. Previous in vitro studies have demonstrated that VMV-infection leads to apoptosis in sheep choroid plexus cells (SCPC) via induction of both intrinsic and extrinsic pathways with subsequent activation of caspases. 3' azido-2',3'-deoxythymidine (AZT) is a potent and selective Human immunodeficiency virus 1 (HIV-1) reverse transcriptase inhibitor, widely used in antiretroviral therapy; however its effects on retrovirus-induced apoptosis are unknown. Using diverse strategies to detect apoptosis, we analysed the broad range effect of AZT treatment on inhibition of VMV-induced apoptosis. First, we found that AZT treatment inhibited the appearance of characteristic apoptotic morphologic changes documented by DAPI staining and oligonucleosomal DNA laddering. Secondly, AZT treatment inhibited caspase cascade and resulted in (i) diminished caspase-3, -8 and -9 activities and (ii) no fluorescein isothiocynate-[VAD]-fluoromethylketone (FITC-VAD-FMK) in situ labelling in VMV-infected cells treated with AZT. Finally, immunocytochemistry indicated that VMV-infection of SCPC induced the subsequent release of apoptosis inducing factor (AIF), whereas AZT treatment inhibited AIF leakage. Consequently, the anti-apoptotic effects of AZT are not restricted, since AZT treatment blocks all the apoptotic pathways induced during VMV-infection.

Atp Binding Cassette Multidrug Transporters Limit the Anti-HIV Activity of Zidovudine and Indinavir in Infected Human Macrophages

To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

Prolonged perinatal AZT administration and early maternal separation: effects on social and emotional behaviour of periadolescent mice

Zidovudine (AZT) is an effective treatment in preventing perinatal transmission of HIV-1; however, a continuous re-evaluation of the risk–benefit ratio of human exposure to this drug is suggested by both clinical and animal studies. The objective of this study was to assess the medium and long-term effects of pre-postnatal AZT treatment on mouse social and emotional behaviour and the possible interactions between AZT exposure and disruptions in the mother–infant relationship. Pregnant CD-1 mice were administered per os with AZT (160 mg/kg) from pregnancy day 10, throughout delivery, to lactation day 10. In half of the litters, the offspring was separated from the mother for 3 h from postnatal days 2 (PND2) to PND14. On PND35, a 30-min social interaction test was performed and corticosterone levels were measured at the end of the session. On PND80, long-term effects of AZT on emotionality were assess by means of an elevated plus-maze. Results indicate that, on PND35, previous AZT exposure affected social behaviour of the experimental subjects, reducing aggressive interactions in males, while decreasing investigative behaviours in females. At adulthood, AZT inhibited exploratory behaviour in the plus-maze while increasing the frequency of risk-assessment postures in male mice. As for maternal deprivation, this early manipulation exerted a pro-aggressive effect in adolescent male mice, deprived subjects being overall characterised by higher activity levels and a deficit in habituation, an effect also observed in the plus-maze. A significant interaction between AZT and maternal deprivation was found for affiliative behaviours. As for corticosterone levels, no AZT effect was found, while maternal deprivation tended to reduce elevations of this hormone in response to stressful stimuli. Overall results from this study indicate that both AZT exposure and maternal deprivation induced gender-dependent changes in social and emotional behaviour both during adolescence and at adulthood.

Maternal immune responses and risk of infant infection with HIV-1 after a short course Zidovudine in a cohort of HIV-1 infected pregnant women in rural Kenya.

To investigate the effects of short-course nucleoside reverse transcriptase inhibitor (Zidovudine, ZDW/AZT) on maternal immune responses and risk of infant infection with HIV-1 among rural-based mothers in western Kenya. A prospective cohort study involving HIV-1 seropositive pregnant mothers and their infants. One hundred and seven HIV-1 seropositive asymptomatic pregnant women and their infants. After informed consent, the women were enrolled at gestation age between 16-24 weeks. For cultural and economic reasons, all mothers were allowed to breast feed their infants. Short-course antepartum regime of AZT was administered to all mothers starting at 36 weeks gestation until start of labour. Maternal absolute CD4+ T cell subset assays were performed before 3rd trimester (about 36 weeks gestation) and after a 4-week therapy of AZT (at least one month post-nuptially). Infant HIV-1 status was determined by HIV-1 DNA polymerase chain reaction (PCR) on samples sequentially taken at 1, 2, 3, 4, 6 and 9 months and confirmed by serology at 18 months of age. Antepartum short-course orally administered AZT: 300mg twice-daily starting at 36 weeks gestation until start of labour, 300mg at labour onset and 300mg every three hours during labour until delivery. Maternal CD4+ T cell counts before and after AZT treatment. Determination of infant HIV-1 infection status. Among 107 women sampled, only 59 received full dose of AZT and thus qualified for present analysis. Of these, 12 infected their children with HIV, while 47 did not. Comparison of CD4+ T cells before and after AZT treatment scored a significant rise in all mothers (P = 0.01). This increase in CD4+ T cells was not significant among mothers who infected their infants with HIV-1 (P = 0.474). However, a significant rise in CD4+ T cells following AZT therapy was observed only in mothers who did not transmit HIV-1 to their infants (P=0.014). These data suggest that a rise in the CD4+ T cell counts following short AZT regimen, now widely in use in resource-weak countries, may be evidence of the active suppression of the replication of HIV. However, further studies to examine the multi-factorial effect of CD4+ lymphocytes and pregnancy on MTCT of HIV need to be carried out to help fully explain the effect of AZT on immune response and whether the CD4+T cell count can be used as a true test of immunological normalisation during antiretroviral therapy.

The effects of perinatal AZT exposure on the acoustic startle response in adult rats

AZT (azidothymidine, zidovudine, ZDV) has become the standard medication to prevent the transmission of the human immunodeficiency virus from mother to fetus. The present study was designed to assess the acoustic startle response (ASR) in adult rats that had been perinatally exposed to AZT. Each litter was randomly assigned to a treatment group: nontreated, AZT 0, 50, 100 or 150 mg/kg. Once daily gastric intubation began prenatally between gestational day (G) 19 and 22 and then continued postnatally between postnatal day (PND) 2 and 20. Between PND75 and PND80, animals were tested for habituation to the acoustic stimuli and prepulse inhibition following a challenge of either saline or 1.0 mg/kg amphetamine (AMP) intraperitoneally. Amphetamine increased ASR and startle latencies throughout the session. The AZT100 dose increased ASR habituation. AZT treatment did not affect prepulse inhibition. Females treated with AZT150 continued to show high ASRs at the end of the startle session. AZT-treated animals showed a dose-dependent increase in peak latency, suggesting a possible abnormal conduction velocity. These effects are independent of handling and intubation effects. Therefore, perinatal AZT treatment results in long-term changes within the primary acoustic startle pathway.

Nucleoside reverse transcriptase inhibitors impair endothelium-dependent relaxation by increasing superoxide.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been used successfully to reduce acquired immunodeficiency syndrome mortality. However, the use of these compounds is associated with numerous tissue toxicities, including cardiomyopathy. These studies address the effects of NRTIs on vascular function. Functional assays of contraction and relaxation were performed on isolated mouse aorta segments obtained from FVB/n mice exposed to zidovudine (AZT), stavudine, or water for 35 days. AZT and stavudine treatment impaired sensitivity to endothelium-dependent relaxation by acetylcholine. Dihydroethidium staining revealed that AZT treatment was associated with an increase in superoxide levels. Pretreatment of AZT-treated vessels with tiron (1 mM), a free radical scavenger, restored endothelium-dependent relaxation in mice. In cellular preparations, electron spin resonance measurements revealed elevated superoxide in cultured endothelial cells exposed to AZT; elevation was dependent on the length of exposure. These results indicate that NRTIs impair endothelium-dependent relaxation by increasing superoxide levels and suggest that NRTI therapy contributes to cardiovascular complications in acquired immunodeficiency syndrome.

Changes of Telomerase Activity and Proliferation by Inhibition of Reverse Transcriptase Activity in Human Cancer Cell.

Activation of telomerase is proposed to be an essential step in cancer cell immortalization and cancer progression. 3'-azido-2',3'-dideoxythymidine (AZT), a reverse transcriptase inhibitor, was reported to be incorporated in telomeric sequences of immortalized cells in culture and to suppress the activity of telomerase and the cell proliferation. In this study, after induction of cancer cell senescence with long-term treatment of AZT, we investigated the dynamics of telomerase subunits (hTERT, hTR, TEP), transcription factors (c-Myc, Mad1), telomerase activity, and finally, telomere length in a human breast cancer cell line. MATERIALS AND METGODS: Human breast cancer cell (MDA-MB-231) was treated with AZT. Senescence was measured by senescence-associated beta-gal staining and apoptosis was counted by dTd enzyme assay. Telomerase activity (by TRAP assay), expression of telomerase subunit genes (by RT-PCR and real-time PCR) and telomere length (by Southern blot analysis) were measured after the AZT treatment. We found evidences of senescence, apoptosis and growth delay after AZT treatment. In addition, AZT- treated cancer cells showed inhibition of telomerase activity and shortening of telomere length in a dose- and duration-dependent way. Among the telomerase subunits, hTERT and c-Myc were the first factors to change after AZT treatment, subsequently, followed by the changes of hTR, Mad1 and TEP. The suppression of hTERT and c-Myc by AZT treatment was the initial genetic phenomenon, subsequently followed by the changes of hTR, Mad1 and TEP.

ZIDOVUDINE-MEDIATED NEONATAL DIAPHRAGM CONTRACTILE DYSFUNCTION

Zidovudine (AZT, 3'-azido-3'deoxythymidine) is a primary drug used to treat HIV/AIDS patients worldwide including the prevention of HIV vertical transmission in pregnant women. While AZT effectively inhibits HIV reverse transcriptase and thus viral replication, studies have also shown AZT to induce a drug-specific muscle myopathy and increased oxidative stress via inhibition of mitochondrial DNA γ polymerase. Previously we have shown that AZT treatment produces respiratory muscle contractile dysfunction in adults rodents (Herb et al., 1997, 2000). To determine if neonatal respiratory muscle was susceptible to AZT toxicity, adult, Sprague-Dawley dams (n = 12) were randomly assigned to a control group or treated with AZT (10mg/kg/day) administered via drinking water ad libitum 28 days prior to and throughout gestation (21 days). Following birth, adult females and pups were maintained on oral AZT for 28 days post-partum prior to contractile experiments. Respiratory muscle function was assessed using two costal diaphragm strips removed from each neonate and evaluated for force-frequency relationship, maximal isometric specific tension, and fatigue resistance (30 min test) using a field stimulation preparation. In vitro studies indicated that neonatal AZT treatment significantly altered the F-F relationship, decreased specific tension development (21%) and reduced fatigue resistance (min 20+) when compared to controls. To our knowledge, this study is the first to address the impact of AZT on neonatal respiratory muscle function. These data support the hypothesis that in utero and post-partum exposure to AZT deleteriously impacts neonatal diaphragm function in a manner similar to that seen in adult diaphragms. We conclude that these data provide evidence indicating the need for an improved understanding of the mechanism(s) responsible for the observed results as well as clinical therapies that can ameliorate the impact of AZT on neonatal respiratory muscle function. Supported by the American Lung Association, NAU Hooper Undergraduate Research Grant, & GlaxoSmithKline

Long-term effects of developmental exposure to zidovudine on exploratory behavior and novelty discrimination in CD-1 mice

Long-term changes in exploratory, social and agonistic behavior have been reported in rodents following developmental exposure to zidovudine (AZT), an agent commonly administered to pregnant seropositive women and their neonates to prevent HIV-1 transmission. The present study evaluates the effects of either prenatal or prolonged AZT treatment on spatial and nonspatial novelty discrimination in mice, using an open-field test with four objects, in which responses to both spatial rearrangement of familiar objects and object novelty are assessed. AZT (160 mg/kg) or Saline was given orally twice daily to pregnant mice from gestational days (GD) 10 to 19 (Experiment 1) or from GD 10 to lactation day 10 (Experiment 2). Offspring of both sexes were tested on postnatal day (PND) 28, 45 or 70. Depending on treatment schedule, AZT altered different behavioral responses, males being more affected than females. The prenatal treatment (Experiment 1) reduced exploration of the objects at all ages considered and increased wall and top rearing at ages 45 and 70. Following prolonged treatment (Experiment 2), AZT offspring were markedly more active than controls and displayed more wall rearing at age 70 while showing lower grooming frequency at all ages. Both AZT and control mice failed to respond to object rearrangement at adulthood, a discrepancy from previous data, which is discussed in relation to perinatal stress effects.

Expression and processing of emotions: Relationships with cd4+ levels in 42 hiv-positive asymptomatic individuals

Background: The study investigated the relationship between CD4+ levels and two emotion-related measures, one of expressed emotion (Hostility directed Inwards, Hdl) and one of capacity for emotional processing (Referential Activity, RA). Method42 HIV-1 positive asymptomatic subjects, under AZT treatment, underwent CD4+ assessment immediately after a brief interview. Interviews were recorded, transcribed and analyzed using the Gottschalk-Gleser and Referential Activity scales. Of the Gottschalk-Gleser scales, only Hdl was considered. ResultsAs hypothesized, the regression of emotion-related scores on CD4 + levels showed a curvilinearrelationship with Hdl scores, and a linearrelationship with RA scores. Subjects with the best immune status therefore showed intermediate levels of expressed emotion and a high capacity for emotional processing. ConclusionThe findings support the usefulness of working through, rather than releasing emotion. Previous negative findings in the field might be explained by the unjustified assumption of linear relationships between expressed emotion and health.

Antiviral resistance of biologic HIV-2 clones obtained from individuals on nucleoside reverse transcriptase inhibitor therapy.

To study phenotypic and genotypic resistance of HIV-2 against nucleoside reverse transcriptase inhibitors (NRTI). Biologic HIV-2 clones were generated from 3 patients before and after initiation of antiretroviral therapy with zidovudine (AZT) in patient RH2-7, AZT and didanosine (ddI) in patient PH2-1, and after addition of lamivudine (3TC) to AZT monotherapy in patient RH2-5. The sensitivity to NRTI of the virus clones, as defined by the 50% inhibitory concentration (IC(50)), was determined in vitro. The predicted amino acid sequences of the reverse transcriptase proteins from these clones were determined. Comparing the sensitivity of the biologic HIV-2 clones obtained after start of therapy with those from antiviral naive patients, resistance had developed to AZT (patients RH2-7 and RH2-5) and 3TC (patient PH2-1 and RH2-5). No resistance to AZT was observed in the biologic clone from PH2-1 obtained after start of therapy. The resistant clones from RH2-5 and PH2-1, but not RH2-7, contained amino acid mutations at positions where HIV-1 has been shown to mutate after AZT and 3TC treatment. Phenotypic resistance of HIV-2 to nucleoside analogues, which developed in HIV-2-infected patients treated with NRTIs, was associated with genotypic changes. Some of the mutations at amino acid positions in the HIV-2 reverse transcriptase gene corresponded with those involved in HIV-1 resistance, although no conventional mutations associated with resistance to AZT were observed.

Evidences that zidovudine (AZT) could not be directly responsible for iron overload in AZT-treated patients: an in vitro study

Zidovudine (3′-azido-3′-deoxythymidine or azidothymidine, AZT) has been the first antiretroviral agent approved for clinical use, and it is still currently used in combination therapy of human immunodeficency virus (HIV) infection. On the basis of increasing clinical reports and in vitro studies, a strict correlation between AZT treatment of HIV positive patients and both the development of anemia and iron overload have been in evidence over the last few years. In this report, we have examined some features of zidovudine to better assess a likely implication of this drug in iron overload. For this purpose, we first determinated the iron chelating ability of both AZT and some of its phosphorylated derivatives in solution. The iron chelating ability of AZT toward the intracellular ‘chelatable’ iron pool was also evaluated. Finally, we investigated the effect of AZT on both iron and transferrin uptake. Our findings indicate that AZT per se cannot be directly responsible for the development of the iron overload found in human or animal models, for which other possible mechanisms are claimed to be involved.

Prolonged perinatal exposure to AZT affects aggressive behaviour of adult CD-1 mice.

AZT is commonly administered to seropositive women and their neonates to prevent mother-to-child transmission of HIV. Recently, animal studies performed in monkeys and rodents have revealed that pre- and/or perinatal exposure to AZT induces age- and sex-dependent behavioural alterations in the offspring, possibly resulting from an action of this drug on CNS targets. Long-term effects of prenatal AZT treatment on social/aggressive behaviour of adult male mice have been previously described. Specifically, AZT has been shown to induce selective changes in the offensive components of agonistic interactions. The aim of the present study was to extend previous findings, analysing the long-term effects of a more prolonged AZT exposure on intraspecific male mice agonistic behaviour. AZT was given orally twice daily to pregnant CD- mice. The dosage selected for AZT was 160 mg/kg. Saline solution (0.9% NaCl) was used as vehicle. Starting on postnatal day (PND) 60 isolated males underwent five 15-min repeated encounters with an opponent of the same age and strain isolated for the same amount of time. Furthermore, a locomotor activity test (PND 67) and a hot-plate test (52 +/- 0.1 degrees C) (PND 74) were performed to assess AZT effects on, respectively, general activity and pain sensitivity. AZT perinatal exposure reduced attack behaviour of adult mice, while increasing the likelihood of them behaving as subordinates. Furthermore, long-term effects of AZT treatment on pain sensitivity were found in the hot-plate test, with AZT mice showing higher pain thresholds than controls. Overall, these data indicate that perinatal exposure to drugs such as AZT exerts selective effects on the developing CNS, resulting in long-term behavioural disturbances. Future studies will need to address the issue of the specific mechanisms underlying these effects.

Effect of zidovudine on the primary cytolytic T-lymphocyte response and T-cell effector function.

Azidothymidine (AZT) and other nucleoside analogues, used to treat AIDS, can cause severe clinical side effects and are suspected of suppressing immune cell proliferation and effector immune cell function. The purpose of the present study was to quantitatively measure the effects of AZT on cytotoxic T-lymphocyte (CTL) priming and to determine if the major histocompatibility complex-restricted CTL killing was affected by AZT exposure. For this purpose, we employed a murine alloantigen model and limiting-dilution analysis (LDA) to estimate cytotoxic effector cell frequencies of alloreactive splenocytes treated with drug during antigen sensitization. This noninfectious model was chosen to avoid analysis of a virus-compromised immune system. Exposure of splenocytes to therapeutic concentrations of AZT (2 to 10 microM) caused a two- to threefold dose-dependent reduction in CLT precursor frequency. This reduction was caused by decreased proliferation of alloantigen-specific CTLs rather than loss of function, because full cytolytic function could be restored by adjusting the AZT-treated effector/target cell ratios to that of untreated cells. In addition, when AZT was added to the assay system at various times during antigen sensitization there was a time-related loss of the suppressive effect on the generation of cytolytic effector function, suggesting that functional CTLs are not affected by even high doses of AZT. Taken together, the data indicate that the reduction of CTL function associated with AZT treatment is due to a quantitative decrease of effector cell precursor frequency rather than to direct drug cytotoxicity or interference with mediation of cytolysis. Furthermore, antigen-naive immune cells were most sensitive to this effect during the first few days following antigen encounter.

Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells.

Numerous studies have reported effects of antiviral nucleoside analogs on mitochondrial function, but they have not correlated well with the observed toxic side effects. By comparing the effects of the five Food and Drug Administration-approved anti-human immunodeficiency virus nucleoside analogs, zidovudine (3'-azido-3'-deoxythymidine) (AZT), 2',3'-dideoxycytidine (ddC), 2', 3'-dideoxyinosine (ddI), 2',3'-didehydro-2',3'-deoxythymidine (d4T), and beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), as well as the metabolite of AZT, 3'-amino-3'-deoxythymidine (AMT), on mitochondrial function in a human hepatoma cell line, this issue has been reexamined. Evidence for a number of mitochondrial defects with AZT, ddC, and ddI was found, but only AZT induced a marked rise in lactic acid levels. Only in mitochondria isolated from AZT (50 microM)-treated cells was significant inhibition of cytochrome c oxidase and citrate synthase found. Our investigations also demonstrated that AZT, d4T, and 3TC did not affect the synthesis of the 11 polypeptides encoded by mitochondrial DNA, while ddC caused 70% reduction of total polypeptide content and ddI reduced by 43% the total content of 8 polypeptides (including NADH dehydrogenase subunits 1, 2, 4, and 5, cytochrome c oxidase subunits I to III, and cytochrome b). We hypothesize that in hepatocytes the reserve capacity for mitochondrial respiration is such that inhibition of respiratory enzymes is unlikely to become critical. In contrast, the combined inhibition of the citric acid cycle and electron transport greatly enhances the dependence of the cell on glycolysis and may explain why apparent mitochondrial dysfunction is more prevalent with AZT treatment.

Cardiac Dysfunction Occurs in the HIV-1 Transgenic Mouse Treated with Zidovudine

Cardiomyopathy in AIDS is an increasingly important clinical problem. Mechanisms of AIDS cardiomyopathy were explored using AIDS transgenic mice that express replication-incompetent HIV-1 (NL4–3Δ gag/pol). Transgenic and FVB/n mice (n = 3 to 6 per cohort) received water ad libitum with and without zidovudine (3′-azido-2′,3′-deoxythymidine; AZT; 0.7 mg/ml) for 21 or 35 days. After 21 days, echocardiographic studies were performed and abundance of mRNA for cardiac sarcoplasmic reticulum calcium ATPase (SERCA2), sodium calcium exchanger (NCX1), and atrial natriuretic factor were determined individually using Northern analysis of extracts of left ventricles. After 35 days, contractile function and relaxation were analyzed in isolated work-performing hearts. Histopathological and ultrastructural (transmission electron microscopy) changes were identified. After 21 days, molecular indicators of cardiac dysfunction were found. Depressed SERCA2 and increased atrial natriuretic factor mRNA abundance occurred in left ventricles from AZT-treated transgenic mice. NCX1 abundance was unchanged. Eccentric left ventricle hypertrophy was determined echocardiographically. After 35 days, cardiac dysfunction was worst in AZT-treated and AZT-untreated transgenic mice. Decreases in the first derivative of the maximal change in left ventricle systolic pressure with respect to time (+dP/dt) occurred in transgenic mice with and without AZT. Increased half-time of relaxation and ventricular relaxation (−dP/dt) occurred in AZT-treated and -untreated transgenic mice. Increased time to peak pressure was found only in AZT-treated transgenic mice. In AZT-treated FVB/n mice, −dP/dt was decreased. Ultrastructurally, mitochondrial destruction was most pronounced in AZT-treated transgenic mice, but also was found in AZT-treated FVB/n mice. Transgenic mice that express HIV-1 demonstrate cardiac dysfunction. AZT treatment of FVB/n mice causes mitochondrial ultrastructural alterations that are similar to those in other species. In transgenic mice, AZT treatment worsens molecular and ultrastructural features of cardiomyopathy. HIV-1 constructs and AZT each contribute to cardiac dysfunction in this murine model of AIDS cardiomyopathy.