In vitro and in vivo toxicity of 2',3'-dideoxycytidine in mice.

3T3 mouse embryo fibroblast cell growth was inhibited in a concentration dependent manner by 2',3'-dideoxycytidine (ddCyd), a strong inhibitor of human immunodeficiency virus. Cell growth inhibition was associated with an increased incorporation of ddCyd into cell DNA. In contrast SP2/0-Ag14 (a mouse myeloma) cell growth is not inhibited by 100 microM ddCyd both in the presence or absence of hypoxanthine and thymidine. Furthermore, in vitro spleen cell proliferation, upon phytohemagglutinin (PHA) addition, was much more affected by ddCyd in C57BL/6 mice than in Swiss albino mice. That indeed ddCyd affects spleen cell proliferation was confirmed by studies on splenocytes obtained from C57BL/6 mice that received ddCyd for 2 weeks in drinking water. These results suggest that ddCyd toxicity in mice is cell and strain dependent and that the toxicity mechanism is related to the incorporation of the drug in cell DNA.     

Cellular metabolism of 2',3'-dideoxycytidine, a compound active against human immunodeficiency virus in vitro

The nucleoside analog 2',3'-dideoxycytidine (ddCyd) has been shown to inhibit the infectivity and cytopathic effect of human immunodeficiency virus on human OKT4+ lymphocytes in vitro. Metabolism of ddCyd by human T-lymphoblastic cells (Molt 4) negative for human immunodeficiency virus and OKT4 was examined. Molt 4 cells accumulated ddCyd and its phosphorylated derivatives into acid-soluble and acid-insoluble material in a dose-dependent manner. For each concentration tested, 2',3'-dideoxycytidine triphosphate represented 40% of the total acid-soluble pool of ddCyd metabolites. Uptake of 5 microM ddCyd was linear for 4 h after addition of drug. Efflux of ddCyd metabolites from cells followed a biphasic course with an initial retention half-life of 2.6 h for 2',3'-dideoxycytidine triphosphate. DNA, but not RNA, of cells incubated with [3H]ddCyd became radiolabeled. Nuclease and phosphatase treatment of DNA followed by reverse-phase high pressure liquid chromatography showed that the nucleoside was incorporated into DNA in its original form. ddCyd was not susceptible to deamination by human Cyd-dCyd deaminase. It was a poor substrate for human cytoplasmic and mitochondrial dCyd kinases, with Km values of 180 +/- 30 and 120 +/- 20 microM, respectively. DNA polymerases alpha, beta, and gamma varied in their sensitivity to inhibition by ddCTP with Ki values of 110 +/- 40, 2.6 +/- 0.3, and 0.016 +/- 0.008 microM, respectively; however, inhibition was competitive with dCTP in each case.     

Human red blood cells as bioreactors for the release of 2',3'-dideoxycytidine, an inhibitor of HIV infectivity

2',3'-Dideoxycytidine (ddCyd) is one of the most potent antiviral nucleosides for killing the human immunodeficiency virus (HIV). ddCyd is currently used in the treatment of severe HIV infections but due to its rapid clearance it must be administered to patients every 4 h reaching concentrations that are toxic. We have synthesized 2',3'-dideoxycytidine-5'-phosphate (ddCMP) as a prodrug, encapsulated it in human erythrocytes and found that it is dephosphorylated by endogenous pyrimidine nucleotidases and subsequently released by the cells as ddCyd. Encapsulated ddCMP does not affect erythrocyte metabolism and was not deaminated by cytidine deaminase. The dephosphorylation reaction has an apparent Km of 6mM, an optimum pH of 6.8 and is not inhibited by ATP or 2,3-bisphosphoglycerate. The efflux of ddCyd from the erythrocyte is a linear function of ddCyd concentration and relatively insensitive to nucleoside transporter inhibitors suggesting that ddCyd permeates the erythrocyte membrane predominantly by nonfacilitated diffusion. Thus, ddCMP-loaded erythrocytes might be used as endogenous bioreactors for ddCyd delivery in the treatment of HIV infection.     

Potent and selective anti-HTLV-III/LAV activity of 2',3'-dideoxycytidinene, the 2',3'-unsaturated derivative of 2',3'-dideoxycytidine

2',3'-Dideoxycytidinene (ddeCyd), the 2',3'-unsaturated derivative of 2',3'-dideoxycytidine (ddCyd) is, like ddCyd itself, a potent and selective inhibitor of HTLV-III/LAV in vitro. This conclusion is based on the relatively high ratio of effective antiviral dose (0.3 microM) versus cell growth inhibitory concentration (20-35 microM) and the lack of any appreciable inhibitory activity against a series of non-oncogenic RNA and DNA viruses. Both compounds were considerably more inhibitory to human lymphoid cell lines than human nonlymphoid or murine cell lines. They were highly dependent on prior activation by deoxycytidine kinase to exert their anti-HTLV-III/LAV and cytostatic effects. In contrast with ddCyd, ddeCyd lost part of its anti-retrovirus effect upon prolonged incubation (10 days) with the virus-infected cells in culture.     

2',3'-dideoxycytidine permeation of the human erythrocyte membrane

The mechanism by which 2,3'-dideoxycytidine, an inhibitor of HIV-I infectivity, permeates the cell membrane was investigated. The influx of ddCyd into human erythrocytes was nonconcentrative. The initial velocity of both ddCyd influx and efflux was, in contrast to compounds that permeate the cell membrane via the nucleoside transporter, a linear function of nucleoside concentration in the 1 microM to 10 mM range and relatively insensitive to temperature. Furthermore, potent inhibitors of nucleoside transporter and other nucleosides were found to inhibit ddCyd influx only partially or not at all suggesting that ddCyd permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic seems to be due to the lack of 3'-hydroxyl moiety of ddCyd which appears to be an important determinant for the nucleoside carrier specificity rather than to lipid solubility itself. As far as permeation of the cell membrane is concerned ddCyd shares these properties with 2',3'-dideoxythymidine and 3'-azido-3'-deoxythymidine.     

Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma

Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K(m) and k(cat) values for insertion of 2',3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2',3'-dideoxy-CTP (ddCTP), 2',3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3' termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.     

Metabolism of 4'-azidothymidine. A compound with potent and selective activity against the human immunodeficiency virus.

4'-Azidothymidine (ADRT) is a novel nucleoside analog, that selectively inhibits human immunodeficiency virus replication in human lymphocytes. Unlike the dideoxyribonucleoside analogs and 3'-azido-2',3'-dideoxythymidine (AZT), ADRT retains the 3'-hydroxy group. The pathways of ADRT metabolism were elucidated by determining: (i) the kinetics of the interactions of ADRT and its metabolites with enzymes of thymidine metabolic pathways, (ii) the pool sizes of phosphorylated metabolites, and (iii) the nature of ADRT incorporation into human DNA. ADRT is not a substrate for thymidine phosphorylase, but is metabolized by kinases. Thymidine kinase phosphorylates ADRT to ADRT monophosphate (ADRT-MP). For this enzyme, ADRT has a Ki value of 5.2 microM, in comparison to a Km value of 0.7 microM for thymidine. The Km value of ADRT toward thymidine kinase is 8.3 microM and the rate of ADRT phosphorylation is 1.4% that of thymidine phosphorylation. ADRT-MP has a low affinity toward thymidylate kinase (a Ki value of 28.9 microM versus a Km value of 0.56 microM for thymidylate), and toward thymidylate synthase (a Ki value of 180 microM versus a Km value of 8 microM for deoxyuridylate). The results suggest that ADRT can be activated effectively by cellular kinases without significant interference of normal thymidine metabolism. In cultured human lymphocytes (A3.01, H9, and U937 cells), ADRT was phosphorylated efficiently to ADRT 5'-triphosphate (ADRT-TP), which is the major metabolite of ADRT. The intracellular concentrations of ADRT-TP ranged from 1 to 3.3 microM after 24 h of incubation with 2 microM of ADRT and the half-life of ADRT-TP varied from 3 to 6 h. Although ADRT-TP is a poor competitive inhibitor against dTTP toward DNA polymerases alpha and beta with Ki values of 62.5 and 150 microM, respectively. ADRT-MP was found to be internally incorporated into cellular DNA. The extent of ADRT-MP substitution for dTMP in DNA was 1 in 6979 for A3.01 cells incubated with 2.9 microM ADRT for 24 h. Internal incorporation of ADRT-MP contrasts with the mechanism of other 2',3'-dideoxynucleoside analogs (i.e. AZT, ddC, ddI, d4T...), which are DNA chain terminators. This finding indicates that a 3'-deoxy structure in a nucleoside analog is not a prerequisite for anti-human immunodeficiency virus activity.     

Both 2',3'-dideoxythymidine and its 2',3'-unsaturated derivative (2',3'-dideoxythymidinene) are potent and selective inhibitors of human immunodeficiency virus replication in vitro

2',3'-Dideoxythymidine (ddThd) and its 2',3'-unsaturated derivative 2',3'-dideoxythymidinene (ddeThd) are potent and selective inhibitors of human immunodeficiency virus (HIV) in vitro. When evaluated for their inhibitory effects on the cytopathogenicity of HIV in MT-4 cells, ddThd and ddeThd completely protected the cells against destruction by the virus at a concentration of 1 microM and 0.04 microM, respectively. In this aspect, ddeThd was about 5 times more potent than 2',3'-dideoxycytidine (ddCyd), one of the most potent and selective anti-HIV compounds now pursued for its therapeutic potential in the treatment of AIDS. ddThd and ddeThd also suppressed HIV antigen expression at 1 microM and 0.04 microM, respectively. Their selectivity indexes, as based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, were 120 (ddeThd) and greater than 625 (ddThd).     

alpha-Tocopherol, but not gamma-tocopherol inhibits 7 beta-hydroxycholesterol-induced apoptosis in human U937 cells.

Oxysterols, particularly those oxidised at position 7, are toxic to cells in culture and have been shown to induce apoptosis in cell types such as vascular endothelial cells, smooth muscle cells and monocytes. The precise mechanism by which oxysterols induce apoptosis is unknown but may involve the generation of oxidative stress. In the present study we examined the ability of alpha-TOC, alpha-TOC acetate (alpha-TOCA) and gamma-TOC to protect against 7 beta-hydroxycholesterol (7 beta-OHC)-induced apoptosis of human monocytic U937 cells. 7 beta-OHC is one of the most commonly detected oxysterols in foods and its level in plasma has been positively associated with an increased risk of atherosclerosis. The present study demonstrates a significant decrease in cell membrane integrity and cellular glutathione levels when U937 cells were treated with 30 microM 7 beta-OHC. DNA fragmentation also occurred, as measured by agarose gel electrophoresis, and the number of apoptotic cells increased as assessed by nuclear morphology. Analysis by HPLC showed that there was a greater incorporation of gamma-TOC into U937 cells after a 48 h incubation, than either alpha-TOC or alpha-TOCA. However, despite the increased uptake of gamma-TOC, only alpha-TOC, and not gamma-TOC or alpha-TOCA was effective at inhibiting 7 beta-OHC-induced apoptosis in U937 cells.     

Cytotoxicity, genotoxicity and oxidative reactions in cell-culture models: modulatory effects of phytochemicals

Much research effort has focused on the identification of phytochemicals in fruit and vegetables which exert beneficial effects. Our research examines modulatory effects of phytochemicals on cytotoxicity, genotoxicity and oxidative reactions in cell systems. Two examples of our studies are discussed. First, the potential beneficial effects of flavonoids are demonstrated. Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free-radical-scavenging activities. The aim of the study was to determine if flavonoids could protect against H2O2-induced DNA damage, as measured by the comet assay, in Caco-2 and HepG2 cells. Both cell lines were supplemented with increasing concentrations of myricetin, quercetin and rutin for 24 h followed by exposure to H2O2 (50 microM) for 30 min. Exposure to H2O2 for 30 min at 37 degrees C resulted in significant DNA damage and pre-incubation with the flavonoids before H2O2 exposure significantly (P <0.05) protected Caco-2 and HepG2 cells against H2O2-induced DNA damage. Secondly, we illustrate the use of cellular models to study oxysterol-induced toxicity. Oxysterols are generated during the cooking and processing of foods and may be produced endogenously by the oxidation of membrane lipids. Recent findings suggest that oxysterols may modulate cytotoxicity by exerting effects on the induction of apoptosis. 7beta-Hydroxycholesterol (7beta-OHC) and 25-hydroxycholesterol, both of which are commonly found in foods, were investigated for their abilities to induce apoptosis in a human monocytic blood cell line, U937, and in the human hepatoma cell line, HepG2 cells. U937 and HepG2 cells were incubated for up to 48 h with 30 microM oxysterol. 7beta-OHC induced apoptosis in U937 cells as measured by non-random DNA fragmentation, condensed and fragmented nuclei, and the generation of hypodiploid cells. In contrast, oxysterols may induce cell death by a different mechanism in the hepatoma cells, possibly by necrosis.     

Pharmacological inhibitors of extracellular signal-regulated protein kinases attenuate the apoptotic action of cisplatin in human myeloid leukemia cells via glutathione-independent reduction in intracellular drug accumulation

It has been reported that inhibition of extracellular signal-regulated protein kinases (ERKs) attenuates the toxicity cisplatin (cis-platinum (II)-diammine dichloride) in some cell types. This response was here investigated using human myeloid leukemia cells. Cisplatin stimulated ERK1/2 phosphorylation and caused apoptosis in U-937 promonocytic cells, an effect which was attenuated by the MEK/ERK inhibitors PD98059 and U0126. While ERK1/2 activation was a general phenomenon, irrespective of the used cell type or antitumour drug, the MEK/ERK inhibitors only reduced cisplatin toxicity in human myeloid cells (THP-1, HL-60 and NB-4), but not in RAW 264.7 mouse macrophages and NRK-52E rat renal tubular cells; and failed to reduce the toxicity etoposide, camptothecin, melphalan and arsenic trioxide, in U-937 cells. U0126 attenuated cisplatin-DNA binding and intracellular peroxide accumulation, which are important regulators of cisplatin toxicity. Although cisplatin decreased the intracellular glutathione (GSH) content, which was restored by U0126, treatments with GSH-ethyl ester and dl-buthionine-(S,R)-sulfoximine revealed that GSH does not regulate cisplatin toxicity in the present experimental conditions. In spite of it, PD98059 and U0126 reduced the intracellular accumulation of cisplatin. These results suggest that GSH-independent modulation of drug transport is a major mechanism explaining the anti-apoptotic action of MEK/ERK inhibitors in cisplatin-treated myeloid cells.     

The role of cytoplasmic deoxycytidine kinase in the mitochondrial effects of the anti-human immunodeficiency virus compound, 2',3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddC) is a potent inhibitor of human immunodeficiency virus replication in vitro and shows beneficial effects in AIDS therapy. The compound inhibits mitochondrial DNA (mtDNA) synthesis at a clinically relevant concentration, which could be responsible for the side effects of ddC observed in the clinic. Thymidine (dThd), one of the substrates of mitochondrial deoxypyrimidine kinase (dPyd kinase), was not able to reverse the mitochondrial toxicity of ddC in CEM cells. Furthermore, the cytoplasmic deoxycytidine kinase (dCyd kinase)-deficient CEM cells were highly resistant to the mitochondrial toxicity of ddC. These data suggest a critical role for cytoplasmic dCyd kinase in the mitochondrial toxicity of ddC. The metabolites of ddC, but not ddC itself, were able to inhibit mtDNA synthesis in isolated mitochondria. The potency of the inhibitory effect was in the order of ddCTP greater than ddCDP greater than ddCMP greater than ddC. The lack of inhibition by ddC of mtDNA synthesis could be due to the inefficient ddC phosphorylation in mitochondria. Although the mitochondrial dPyd kinase was reported to phosphorylate ddC, the phosphorylation of ddC in isolated mitochondria was not detectable. The data suggest that ddC is phosphorylated to ddCTP in the cytoplasm and then transported into mitochondria to exert its inhibitory effect on mtDNA synthesis.     

Molecular insights into NRTI inhibition and mitochondrial toxicity revealed from a structural model of the human mitochondrial DNA polymerase

NRTI-based therapy used to treat AIDS can cause mitochondrial toxicity resulting from the incorporation of NRTIs into mitochondrial DNA by DNA polymerase gamma (pol gamma). Pol gamma has poor discrimination against many of the currently used NRTIs resulting in aborted DNA synthesis and subsequent depletion of mtDNA. Pol gamma readily incorporates ddCTP, ddITP and D4T-TP with an efficiency similar to the incorporation of normal nucleotides, whereas AZT-TP, CBV-TP, 3TC-TP and PMPApp act as moderate inhibitors to DNA synthesis. We have sought a structural explanation for the unique selection for NRTIs by the human pol gamma. A structural model of the human pol gamma was developed to ascertain the role of active site amino acids. One residue in particular, Y951 in motif B, is primarily responsible for the selection of dideoxynucleotides and D4T-TP. Our structural model of the human pol gamma should assist in rational design of antiviral nucleoside analogs with higher specificity for HIV-RT and minimal selection and incorporation into mitochondrial DNA.     

Differential effects of ETYA, a PUFA-analogue, on prostate (PC3) and monoblastoid U937 ultrastructure; lack of correlation with reduced proliferation.

ETYA (5,8,11,14-eicosatetraynoic acid), a polyunsaturated fatty acid analogue, inhibits proliferation of PC3 and U937 cells and induces a limited differentiation in U937 cells. Human prostate PC3 cells cultured for 72 h with 40 microM ETYA in fetal calf serum contained putative lipofuscin bodies, myelin figures and mitochondria with damaged cristae and matrices. These changes were absent from human U937 monoblastoid cells incubated with ETYA in CPSR3, a semipurified serum replacement. U937 cells cultured with ETYA in fetal calf serum contained occasional lipofuscin bodies, while PC3 cells cultured in CPSR3 exhibited all of the changes described. ETYA reduced the oxygen consumption of both cell lines. Therefore we conclude: (a) The response to ETYA by cells of dissimilar developmental origin is not identical; (b) unidentified serum components can augment potential ETYA-induced oxidative stress-responses of cells; (c) inhibition of U937 proliferation by ETYA does not depend upon the morphologic changes seen in PC3 cells, which resemble sequelae of oxidative stress with excess free radicals; and (d) rapid ETYA-induced inhibition of oxygen consumption in both cell lines implies a reduced synthesis of ATP that could contribute to the reversible impairment of cellular proliferation.     

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.     

Specific inhibition of DNA biosynthesis induced by 3'-amino-2',3'-dideoxycytidine

3'-Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) produced an S-phase-specific block in exponentially growing L1210 leukemia cells. The monophosphate and triphosphate forms of the drug were detected within a few hours of 3'-NH2-dCyd treatment of intact cells. No significant change in the deoxynucleoside triphosphate levels was observed during the early stages of treatment. However, by 24 h a 2-fold increase in the amount of the deoxynucleoside triphosphates was seen. The triphosphate form of the drug competitively inhibited dCTP incorporation into calf thymus DNA using highly purified DNA polymerase alpha. The Ki was determined to be 9.6 microM with respect to dCTP. Incorporation of the analogue into DNA was not detected. On the other hand, sucrose gradient analysis suggested that incorporation of the analogue into actively synthesized DNA may account for the biological activity of this compound. Treatment with 3'-NH2-dCyd induced single-strand breaks in actively synthesized DNA, but no double-strand breaks were observed in the presence of the analogue. The data indicate that 3'-amino-2',3'-dideoxycytidine specifically interferes with DNA replication at the level of DNA polymerase by inhibiting chain elongation.