Remarkable improvement of neuropsychiatric symptoms in HIV-infected patients after AZT therapy

Summary Treatment of neuropsychiatric symptoms with AZT (Azidothymidine) seems to be effective in many AIDS patients. In earlier studies, 204 of 525 patients treated with AZT showed neuropsychiatric improvement. In this presentation, two patients with affective disorder and dementia are described. Both patients showed remarkable improvement of their severe psychiatric symptoms after AZT. The authors recommend treatment of AIDS-associated dementia and psychoorganic symptoms with AZT.Abbreviations AZT azidothymidine - CT computer tomography - CSF cerebrospinal fluid - MRI magnetic resonance imaging - PET positron emission tomography Supported by a grant of the German Federal Government III-02/89     

Chronic In Vitro Exposure to 3′-Azido-2′, 3′-Dideoxythymidine Induces Senescence and Apoptosis and Reduces Tumorigenicity of Metastatic Mouse Mammary Tumor Cells

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

Human inter-individual variability in metabolism and genotoxic response to zidovudine

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.     

Pre-bled-young-rats in genotoxicity testing: a model for peripheral blood micronucleus assay

Previously we have reported that prior bleeding increases the sensitivity of micronucleus (MN) assay in rats (Vikram et al., 2007 a). Rat peripheral blood micronucleus (PBMN) assay is generally not considered as reliable method for the assessment of clastogenic potential of test chemicals due to selective elimination of micronucleated cells from the circulation. The present study is aimed to evaluate the sensitivity of pre-bled-young-rat model in detecting genotoxins having different mechanism of action. In the present study, young male Sprague-Dawley (SD) rats (21-24 days old, weighing 60+/-5 g) and swiss mice (24-28 days old, weighing 15+/-2g) were used. Streptozotocin (STZ, 50mg/kg), Methotrexate (MTX, 10mg/kg), N-nitrosodiethylamine (DEN, 200mg/kg), Quercetin (QC, 50mg/kg) and Zidovudine (AZT, 400mg/kg) were used in the present experiment. Effect of prior bleeding time (0, 2, 6, 12 and 24h) on the kinetics of MN formation with STZ and AZT was studied and 36 h post chemical exposure was found to be the most suitable time point for sample collection if prior bleeding time was 0, 2 and 6h. Further, the impact of prior bleeding (2h) on the kinetics of MN formation in the bone marrow was evaluated with STZ and maximum MN frequency was observed after 24h. The area under curve (AUC) analysis proves that prior bleeding leads to significant increase in the incidence of micronucleated reticulocytes (RETs) in the peripheral blood as compared to respective non-bled controls. Out of five tested chemicals AZT and STZ induced significant increase in the MN frequency in non-bled animals while at the same dose MTX, AZT, QC and STZ induced significant increase in MN frequency in the pre-bled-young-rats employing PBMN assay as the end point. Positive results with MTX, AZT, QC, STZ and negative results with DEN demonstrate both the sensitivity and specificity of pre-bled-young-rat model in the screening of chemicals for genotoxicity using PBMN assay as the end point.     

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.     

The Development of a Q-Sort Behavioral Rating Procedure for Pediatric HIV Patients

Developed a Q-sort procedure to assess social, emotional, andmotivational behavior associated with central nervous systemdisease among 180 HIV-infected pediatric patients. These ratingswere factor analyzed and scales were derived based on the factorstructure. Younger (M age = 1.03 years) patients with HIV-associatedencephalopathy were rated as more apathetic and nonsocial intheir behavior than nonencephalopathic younger patients. Older(M age = 7.8 years) encephalopathic patients had significantlyhigher scores on scales measuring depression, autism, and irritabilitycompared to nonencephalopathic patients from this age group.A subgroup (26 patients) showed a significant decrease in theseelevated scores after a 6-month course of AZT     

T activation marker evaluation in ARC patients treated with AZT. Comparison with CD4+ lymphocyte count in non-progressors and progressors towards AIDS.

Reductions in the percentage and absolute number of CD4+ lymphocytes, as well as abnormally high levels of activated peripheral T lymphocytes (CD3+ HLA-DR+ phenotype) and an increased proportion of CD8+ cells coexpressing the CD57 surface antigen (involved in natural killer activity) have been reported in HIV infection and associated with disease progression. We prospectively measured these subsets of lymphocytes in 34 patients with advanced AIDS-related complex (ARC) treated with azidothymidine (AZT). Peripheral blood lymphocyte phenotyping was performed before treatment, then at weeks 12 and 24. A striking fall in the proportion of activated T lymphocytes from baseline was observed (P less than 0.001) at week 24. In contrast, the percentage of CD4+ cells showed a slight and transient rise at week 12 (P less than 0.05). No modification in levels of CD8+ or CD8+ CD57+ cells was detected during the study. Of the 34 patients, 11 developed AIDS, and 23 remained AIDS-free during 51 weeks of follow-up. Similar patterns of change in CD4+ and HLA-DR+ CD3+ lymphocytes were found in the AIDS progressors and nonprogressors. Likewise, HIV p24 antigenaemia showed parallel decreases in both groups of patients. Although changes in CD4+ cells, p24 antigenaemia and HLA-DR-reactive T lymphocytes were not predictive of clinical outcome, large differences existed between the two groups prior to the initiation of therapy. The short-term onset of AIDS was associated with lower CD4+ cell numbers, higher levels of serum p24 antigen and a greater proportion of activated T lymphocytes. Our results suggest that the possible interest of T lymphocyte activation markers, in conjunction with conventional phenotyping, should be investigated further.     

AZT对肝癌细胞端粒酶活性及相关蛋白表达的影响

背景与目的：端粒酶抑制剂抑制端粒酶活性的机制十分复杂,可能涉及多个蛋白的共同作用。本研究通过3’-叠氮脱氧胸苷（3’-azido-deoxythymidine,AZT）作用于人肝癌细胞SMMC-7721。比较AZT作用后癌细胞端粒酶活性及相关蛋白的变化,探讨AZT抑制端粒酶活性的可能机制。方法：利用噻唑蓝[3（4,5-demethyhhiazole-2-y1）-2,5-diphenyl tetrazolium-bromide,MTT]实验确定AZT作用的最佳作用时间和浓度;实时荧光定量端粒重复序列扩增法（real-time fluorescent quantitative TRAP assay,FQ-TRAP法）检测AZT作用后,SMMC-7721细胞端粒酶活性变化;用缺口末端标记技术（terminal deoxyribonucleotidyl transferse（TdT）-mediated biotin-16-dUTP nick-end labelling,TUNEL）法和流式细胞术检测AZT作用后．SMMC-7721细胞凋亡情况;单细胞激光拉曼光谱技术和蛋白质芯片-飞行时间质谱仪,检测AZT作用后特异蛋白质的变化。结果：AZT抑制癌细胞生长的最佳作用时间为48h,最佳作用浓度为20mmol／L;FQ-TRAP法检测发现,AZT作用后肝癌细胞端粒酶活性与对照组相比受到明显抑制（P=0．0001）;TUNEL法观察到AZT诱导肝癌细胞凋亡形态学改变．流式细胞术检测AZT诱导肝癌细胞凋亡率为13．5％：单细胞激光拉曼光谱技术观察到,AZT作用后有7个与蛋白代谢相关的特征峰变化;蛋白质芯片．飞行时间质谱仪检测发现,AZT作用后有24个蛋白分子在癌细胞中高表达,8个蛋白分子在癌细胞中低表达,32个差异蛋白均属于小分子蛋白。结论：AZT可抑制SMMC-7721细胞端粒酶活性,诱导凋亡与相关特异小分子蛋白有关。