Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver

After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase-mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase gamma-mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [(3)H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [(3)H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis.     

Zidovudine (AZT) for treatment of patients infected with human immunodeficiency virus type 1. An evaluation of effectiveness in clinical practice

To learn about the patterns of use and the effectiveness of zidovudine therapy in clinical practice, we conducted an observational cohort study of 86 patients with human immunodeficiency virus type 1 infection. All patients were followed up for at least 6 months after starting zidovudine (AZT) therapy. Of the 86 patients, 78 (91%) initially received full-dosage zidovudine (1200 mg/d), and eight received a reduced dosage (600 mg/d). During follow-up, the number able to maintain full-dosage zidovudine therapy decreased to 54 (63%) at 3 months and 40 (47%) at 6 months. Thirty-five patients required dosage reductions that lasted at least 7 days and were not preceded by an adverse outcome (death or opportunistic infection). Overall, adverse outcomes occurred for nine (26%) of those with dosage reductions compared with 22 (43%) of 51 patients with no previous dosage change. Even after adjusting for baseline cytopenias and the time of the dosage reductions, adverse outcomes did not occur significantly more often in patients who received reductions in their zidovudine dosage. Our results indicate that full-dosage zidovudine therapy cannot be maintained for most patients infected with human immunodeficiency virus, but that clinicians need not be pessimistic about treatment outcomes when dosage reductions are needed.     

In vivo prevalence of azidothymidine (AZT) resistance mutations in an AIDS patient before and after AZT therapy

In order to examine the in vivo prevalence of AZT resistance mutations in AID patients after long-term therapy we amplified, by polymerase chain reaction (PCR), a 654 bp pol gene fragment from peripheral blood mononuclear cell DNA samples from a patient before, and 19 months after, the start of AZT therapy. PCR products from each sample were cloned and 9 clones from each sample were sequenced. Seven of 9 clones from the post-AZT sample, but none from the pre-AZT sample, contained an amino acid substitution (Thr215 to Tyr) requiring two nucleotide changes within the same codon (ACC to TAC). This change had previously been shown by Larder and Kemp (Science, 246:1155-1158, 1989) to correlate with partial AZT resistance of virus isolates. In colony hybridizations using synthetic oligonucleotides corresponding to the mutant and wild-type sequences, 22 of 22 clones from the pre-AZT sample hybridized only to the wild-type probe while 21 of 26 clones from the post-AZT sample hybridized only to the mutant. Clinically, this patient remains well, indicating that while Tyr215 may be the first amino acid substitution leading to resistance, it alone does not appear to have significantly influenced the clinical status of this patient.     

Absence of peroxisomes in mouse hepatocytes causes mitochondrial and ER abnormalities

Peroxisome deficiency in men causes severe pathology in several organs, particularly in the brain and liver, but it is still unknown how metabolic abnormalities trigger these defects. In the present study, a mouse model with hepatocyte-selective elimination of peroxisomes was generated by inbreeding Pex5-loxP and albumin-Cre mice to investigate the consequences of peroxisome deletion on the functioning of hepatocytes. Besides the absence of catalase-positive peroxisomes, multiple ultrastructural alterations were noticed, including hepatocyte hypertrophy and hyperplasia, smooth endoplasmic reticulum proliferation, and accumulation of lipid droplets and lysosomes. Most prominent was the abnormal structure of the inner mitochondrial membrane, which bore some similarities with changes observed in Zellweger patients. This was accompanied by severely reduced activities of complex I, III, and V and a collapse of the mitochondrial inner membrane potential. Surprisingly, these abnormalities provoked no significant disturbances of adenosine triphosphate (ATP) levels and redox state of the liver. However, a compensatory increase of glycolysis as an alternative source of ATP and mitochondrial proliferation were observed. No evidence of oxidative damage to proteins or lipids nor elevation of oxidative stress defence mechanisms were found. Altered expression of peroxisome proliferator-activated receptor alpha (PPAR-alpha) regulated genes indicated that PPAR-alpha is activated in the peroxisome-deficient cells. In conclusion, the absence of peroxisomes from mouse hepatocytes has an impact on several other subcellular compartments and metabolic pathways but is not detrimental to the function of the liver parenchyma. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).     

Azidothymidine(AZT)-induced siderosis

Azidothymidine(AZT) interferes with heme synthesis. This should upregulate the synthesis of transferrin receptors and increase the amount of iron taken up by the cell. We found a 50% increase in the iron content of liver and a 20% increase in the iron content of macrophages in AZT-treated mice.     

HIV-associated myocarditis treated with zidovudine (AZT)

Congestive cardiomyopathy has been reported in patients infected with human immunodeficiency virus (HIV). In this report of diffuse lymphocytic myocarditis in a patient who tested positive for HIV antibody after receiving blood from an HIV-positive donor, the patient failed to respond to conventional medical therapy. Although no opportunistic infection or malignancy was observed by electron-and light-microscopic examination of the endomyocardial tissue sample at biopsy, it was decided to give the patient zidovudine (AZT) at a dosage of 200 mg every 4 hours. Definite clinical improvement was noted on repeat right heart catheterization and endomyocardial biopsy 3 months after the initiation of AZT therapy. This case supports the theory that myocarditis can be caused by HIV and suggests that AZT may be useful in the treatment of patients with HIV-associated cardiomyopathy.     

Transfer of zidovudine (AZT) by human placenta

The transfer of zidovudine (AZT) across human placenta was studied using an in vitro perfusion system with independent maternal and fetal circulations. AZT is transferred toward the fetus more rapidly than L-glucose (transfer index 1.5), a water-soluble molecule smaller than AZT (267 vs. 180 Da) that passively diffuses across the placenta. The transfer rate is proportional to the concentration in the maternal perfusate over a range of 0.03-300 microM. Transfer rate in the reverse direction, toward the maternal perfusate, also exceeds that of L-glucose and fails to show saturability. These observations are consistent with simple diffusion. The partition of AZT and glucose between perfusion buffer and octanol is 1.04 and 0.013, respectively, indicating that AZT is more lipophilic and providing a reasonable explanation for the more rapid transfer. AZT is extensively metabolized by the placenta to more polar and as yet unidentified metabolites that are not released into the perfusate. The possibility that the placental accumulation of such metabolite(s) may exert an antiviral action and may also affect placental function must be considered.     

Replication of total and mitochondrial DNA in rat liver with aging

Replication of total (t-DNA) and mitochondrial (mt-DNA) liver DNA of adult (6 months) and old (24 months) female rats labelled with i.v. ³H-thymidine has beeb studied. The most part of t-DNA radioactivity was shown to result from the replication of nuclear DNA (n-DNA). 10, 30, 70 and 120 min after labelling, intracellular acid-soluble ³H-radioactivity of old rats was significantly higher than that of adult ones. This may be due to the change in precursors concentration with aging. Thus, despite the lack of differences between the specific radioactivities of t-DNA and mt-DNA of adult and old animals during all labelling periods used the true replication rate, i.e., specific DNA radioactivity/volume of intracellular pool of label (relative DNA radioactivity), was significantly higher in adult animals during all labelling periods.Decrease of n-DNA and mt-DNA replication rate may lead to lowering of energetic potential and cell renewal in the liver with aging.     

Downregulation of mitochondrial lon protease impairs mitochondrial function and causes hepatic insulin resistance in human liver SK-HEP-1 cells

Aims/hypothesis  

Lon protease degrades oxidatively damaged proteins in mitochondrial matrix. To examine the relationships between mitochondrial quality control, mitochondrial functions and diabetes, we investigated whether lon protease deficiency influences insulin resistance by affecting mitochondrial function.     

Cardiac pathologic effects of azidothymidine (AZT) in Mg-deficient mice

Treatment of HIV with AZT (zidovudine) may have toxic side effects as a result of multiple mechanisms. It is known that patients with AIDS may suffer from magnesium deficiency (MgD). We studied selected biochemical and histopathologic consequences of AZT administration (0.7 mg/mL in drinking water) with concurrent Mg-deficient (20% of normal) diet in male C57Bl/6N mice for 3 wk. Significant decreases in red blood cell glutathione (GSH) were evident in the Mg-deficient mice with or without AZT treatment, suggesting compromised antioxidant capacity in the blood. Although MgD alone led to a 1.9-fold increase in plasma thromboxane B(2) (TXB(2), derived from the highly vasoconstrictive TXA(2)), AZT + MgD increased the TXB(2) level 3.5-fold. AZT (+/-MgD) provoked prominent hepatic damage expressed by distortion of lobular architecture, nuclear and cellular swelling, and inflammatory lesions and loss of hepatocytes. AZT alone caused mild cardiac lesions, resulting in partial cardiac fibrosis, especially in the atrium. AZT + MgD caused only scattered small-size cardiac lesions consisting of microscopic foci of inflammatory infiltrates in the ventricles but led to more prominent lesions, fibrosis, and scars in the atrium. MgD or AZT alone caused varying degrees of skeletal muscle degeneration; in combination, more intense degeneration and regeneration of muscle cells were evident. In conclusion, it is suggested that both the decreased blood GSH and elevated plasma TXA(2) might contribute, at least in part, to the aggravated pathological damages observed in the atrium and skeletal muscle of the AZT-treated Mg-deficient mice.     

Prevalence of mitochondrial DNA haplogroups in an Australian population

Mitochondrial DNA (mtDNA) haplogroups are 'neutral polymorphisms' in the mtDNA genome, which have accumulated and persisted along maternal lineages as the human population has migrated worldwide. Three ethnically distinct lineages of human mtDNA populations have been identified: European, characterized by nine haplogroups H, I, J, K, T, U, V, W and X; African, characterized by superhaplogroup L and Asian, characterized by superhaplogroup M. We studied the prevalence of mtDNA haplogroups in participants of the Blue Mountains Eye Study, a large population-based survey of vision conducted between 1991 and 2000 of non-institutionalized permanent residents aged 49 years or older from two suburban postcode areas, west of Sydney, Australia. Total DNA isolated from either hair follicles or blood was available for 3377 of the 3509 participants (96.2%) to determine mtDNA haplogroups by polymerase chain reaction/restriction fragment length polymorphism analysis. Approximately 94.2% of samples could be assigned to one of the nine major European haplogroups, whereas a further 1.2% included the African (L) and Asian (M) superhaplogroups. The five principal haplogroups represented were H (42.9%), U (14.1%), J (10.7%), T (9.2%) and K (8.1%), which together included 85% of this population.