DNA strand cleavage in vitro by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]-indole, a direct-acting mutagen formed in the metabolism of carcinogenic 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

3-Hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) is a direct-acting mutagenic compound derived by metabolic activation from 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a strongly mutagenic carcinogen. The action of N-OH-Trp-P-2 on DNA in vitro was investigated. N-OH-Trp-P-2 inactivated Bacillus subtilis transforming DNA and produced single-strand cuts in a supercoiled circular DNA (phi X174RFI) under neutral conditions. When mouse FM3A cells in culture were treated with a noncytotoxic dose of N-OH-Trp-P-2 and then the cellular DNA was examined by the alkaline elution technique, chain cleavages of the DNA were observed. Cysteamine inhibited the spontaneous degradation of N-OH-Trp-P-2 and enhanced the covalent binding of [3H]N-OH-Trp-P-2 to DNA. This finding offered an explanation for the previously observed enhancement of Trp-P-2 mutagenicity by cysteamine. In contrast cysteamine inhibited the N-OH-Trp-P-2-mediated inactivation of B. subtilis DNA as well as the strand cleavage in phi X174RFI DNA. The cleavage in phi X174RFI DNA was also inhibited by catalase. These observations indicate that the mutagenicity and DNA-cleaving activity of N-OH-Trp-P-2 are distinct from each other, that the inactivation of transforming DNA was caused mainly by strand cleavage, and that the DNA cleavage was probably caused by active oxygen radicals produced in the oxidative degradation of N-OH-Trp-P-2.

Imbalance of deoxyribonucleoside triphosphates, DNA double-strand breaks, and cell death caused by 2-chlorodeoxyadenosine in mouse FM3A cells.

The mechanism of the cytotoxic action of 2-chlorodeoxyadenosine in mouse FM3A cells was investigated. Imbalance of the dNTP pools occurred within 3 h of treatment with 20 microM 2-chlorodeoxyadenosine; the dATP and dGTP pools were depleted and the dTTP pool increased. 2-Chlorodeoxyadenosine added to the culture medium broke mature DNA strands, giving fragments of 100-200 kilobase pairs as found by orthogonal-field-alternation gel electrophoresis. DNA strand breaks, measured by this technique, were observed in the treated cells about 12 h after the addition. The cells also lost viability at about 12 h. Breaks in the single and double strands of DNA, as measured by alkaline and neutral filter elution, became evident 18 h after treatment with 20 microM 2-chlorodeoxyadenosine; there were as many single-strand breaks as would be caused by 130 rads of gamma-ray irradiation. Double-strand breaks were equivalent to those caused by 2180 rads of gamma-ray irradiation. Comparison of the ratio of single- and double-strand breaks caused by 2-chlorodeoxyadenosine to that following radiation suggested that 2-chlorodeoxyadenosine broke only double strands. Cycloheximide inhibited the breakage of DNA double strands and the cell death caused by this compound. Flow cytometric studies of cytostasis brought about by 2-chlorodeoxyadenosine in FM3A cells showed that cells accumulated in the earlier part of the S phase. 2-Chlorodeoxyadenosine decreased DNA synthesis more than RNA or protein synthesis. The breaks in double strands of DNA were probably important in the cell death caused by 2-chlorodeoxyadenosine. The intracellular dNTP imbalance may trigger these events.

1,2,4,5-Tetraoxacycloalkanes: synthesis and antimalarial activity.

In this short review the methods of preparation of novel 1,2,4,5-tetraoxacycloalkanes and the related peroxides are summarized, with the emphasis on the usefulness of 1,1-bishydroperoxides as the precursor. Also, their antimalarial activities in vitro and in vivo are discussed.

Interaction of 1-(5-phospho-beta-D-arabinofuranosyl)-5-substituted-uracils with thymidylate synthetase: mechanism-based inhibition by 1-(5-phospho-beta-D-arabinosyl)-5-fluorouracil.

A number of 1-(5-phospho-beta-D-arabinosyl)-5-substituted-uracils (ara-UMP's) have been examined as inhibitors of dTMP synthetase. As reversible inhibitors, all were substantially less potent than their 2'-deoxyribosyl counterparts. In the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate), ara-FUMP caused a first-order, time-dependent inactivation of the enzyme. At 0 degrees C, kinetic studies indicated a reversible Kd of 3.6 micro M for the ara-FUMP-CH2-H4folate complex, and k = 0.22 min-1 for the subsequent inactivation. Spectral studies of the complex and its behavior toward protein denaturants demonstrate that its structure and stoichiometry are directly analogous to those which have previously been described for FdUMP. The significance of this finding with regard to prodrugs of ara-FU and the potential of ara-FU as a chemotherapeutic agent are discussed.

Synthesis and antitumor activity of tropolone derivatives. 7. Bistropolones containing connecting methylene chains.

Bistropolone derivatives (4-12) containing differing lengths of linkage between the two tropolone rings were prepared and examined for their antitumor activity in in vitro (KB cell) and in vivo (leukemia P388 in mice) systems. Parent compound 3, related compounds previously prepared, and the new compounds 4-12 were evaluated for inhibitory activity against ribonucleotide reductase by indirect means to measure their effects on the dNTP pool imbalance. Present structure-activity relationship results would suggest that potently active bistropolones in vivo inhibit intracellular ribonucleotide reductase through chelating with the two irons at the two active sites of the enzyme.

Thymidine phosphorylase. Substrate specificity for 5-substituted 2'-deoxyuridines.

A continuous spectrophotometric assay for thymidine phosphorylase has been developed and used to analyze the substrate properties of a number of 5-substituted 2'-deoxyuridines. A QSAR was found which relates the catalytic efficiency for various substrates to a single variable, the inductive field constant F.

Synthesis and antimalarial activity of cyclic peroxides, 1,2,4,5, 7-pentoxocanes and 1,2,4,5-tetroxanes.

A variety of 1,2,4,5,7-pentoxocane and 1,2,4,5-tetroxane derivatives were prepared as potential peroxide antimalarial agents. In both series of cyclic peroxides, the steric and electronic effects of the substituents attached to the peroxide ring exert a remarkable influence on the antimalarial activity. For some cyclic peroxides, which were found to be highly effective in vitro, the study in vivo has been also conducted.

Synthesis and antimalarial activity of novel medium-sized 1,2,4,5-tetraoxacycloalkanes.

CsOH- or Ag(2)O-mediated cycloalkylation of (alkylidene)bisperoxides 3 and 1,n-dihaloalkanes (n = 3-8) provided the corresponding medium-sized 1,2,4,5-tetraoxacycloalkanes 4-8 in moderate yields. Subsequent evaluation of the antimalarial activity of the cyclic peroxides 4-8 in vitro and in vivo revealed that 1,2,6,7-tetraoxaspiro[7.11]nonadecane 4a has considerable potential as a new, inexpensive, and potent antimalarial drug.

New type of febrifugine analogues, bearing a quinolizidine moiety, show potent antimalarial activity against Plasmodium malaria parasite.

Febrifugine (1) and isofebrifugine (2), isolated from the roots of Dichroa febrifuga Lour. (Chinese name: Cháng Shan), are active principles against malaria. Adducts of 1 and 2 with acetone, Df-1 (3) and Df-2 (4), respectively, were obtained using silica gel and acetone. They showed high activity against P. falciparum malaria in vitro. Compound 3 was found to be equally effective against P. berghei in vivo as the clinically used drug chloroquine, whereas 4 showed only 1/24 of the activity of 3. Metabolism studies of these compounds revealed that compound 4 is readily metabolized in mouse liver. Accordingly, the dose of 4 must be higher than that of 3 to attain blood levels sufficient for a favorable therapeutic effect.

Use of blue cotton for detection of mutagenicity in human feces excreted after ingestion of cooked meat.

Fried ground beef has been shown to contain mutagens, and the major mutagenic component has been identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Mutagens in feces of three adult volunteers were fractionated by treatment of the feces with blue cotton followed by chromatography on a carboxymethyl cellulose column. The chromatographic fraction corresponding to MeIQx in terms of the position of elution was examined for mutagenicity in S. typhimurium TA 98 with metabolic activation. When meals containing no heated meat were eaten, this fraction of feces showed little or no mutagenicity. On eating fried ground beef, the feces excreted in the next 2 days showed greatly increased mutagenicity in this fraction. By eating no-meat meals subsequent to the meat meal, the mutagenicity resumed the original low level on the fourth day after the meat meal. The components in the mutagenic fraction were analyzed by high-pressure liquid chromatography, and were shown to differ from MeIQx.

Defective DNA replication and repair associated with decreases in deoxyribonucleotide pools in a mouse cell mutant with thermolabile ubiquitin-activating enzyme E1.

Upon shift-up in temperature, mouse tsFS20 mutant cells with thermolabile ubiquitin-activating enzyme E1 immediately stopped DNA replication and showed cell cycle arrest in S-phase. In contrast, when the cells were permeabilized with lysolecithin after culture at the nonpermissive temperature, they exhibited a normal level of replicative DNA synthesis in vitro. In agreement with this, intracellular pools of deoxyribonucleoside triphosphates were significantly reduced in the cells cultured at the nonpermissive temperature. Even under the permissive conditions, tsFS20 cells were more sensitive to hydroxyurea and alkylating agents, and induced less mutation than the wild-type cells. These results suggest that the ubiquitin system affects DNA replication and repair.

Contamination by ATP of commercial dATP samples causing erroneous results in studies of DNA replication in isolated HeLa cell nuclei.

dATP at high concentrations was capable of replacing ATP required in the synthesis of Okazaki pieces in isolated HeLa cell nuclei. In addition, the levels of synthesis of high molecular weight DNA were observed to vary depending on the lot of dATP used. Analysis by HPLC revealed that dATP samples of a particular source contained ATP in the range of 0.25-0.43 mol%. With ATP-free dATP, almost no synthesis of high molecular weight DNA was observed, while with impure dATP, a small but significant amount of high molecular weight DNA was synthesized. While this observation confirmed our previous finding that dATP can replace ATP in the synthesis of Okazaki pieces but not in the joining of the pieces, it is also a warning to users of commercial dATP in biochemical and biological studies.

Detection of Plasmodium falciparum in human blood by a nested polymerase chain reaction.

We have developed a simple method for direct detection of Plasmodium falciparum parasites in infected human blood using a nested polymerase chain reaction. Whole blood (10 microliters) was obtained by finger puncture and suspended in a microcentrifuge tube containing phosphate-buffered saline. For removal of components that might inhibit the PCR, blood samples were treated with saponin and washed by centrifugation. After three cycles of a two-step incubation (3 min at 94 degrees C and 3 min at 55 degrees C), the first amplification was done with oligonucleotide primers specific for the junction region of the gene coding for the dihydrofolate reductase-thymidylate synthase in P. falciparum. A 1-microliter portion of the first amplification was then amplified again with a second set of primers, and 226-basepair fragments were generated. The amplified products were analyzed by agarose gel electrophoresis with ethidium bromide staining. Experiments with cultured parasites showed that the method could detect as few as 13 parasites in 10 microliters of whole blood. In 1991, 101 samples from 98 donors were collected in Guadalcanal, Solomon Islands. Eight of these samples gave positive results by both examination of thin blood smears and by the nested PCR. There was a correlation between parasite densities and the intensity of the results by the nested PCR. The method is suitable for detection of asymptomatic parasite carriers and evaluation of medical treatment on clinical cases.

A trial for a DNA diagnosis of Plasmodium vivax malaria recently reemerging in the Republic of Korea using microtiter plate hybridization assay.

The polymerase chain reaction-based microtiter plate hybridization (PCR-MPH) assay was utilized for a DNA diagnosis of Plasmodium vivax malaria, which has recently reemerged in the Republic of Korea. The subjects were 18 parasite-proven patients and 5 healthy controls. Follow-up blood samples were collected from 4 patients after a standard course of treatment. Polymerase chain reaction and electrophoresis of all the patients' blood showed a prominent band at the 138 base pair area, but not in the controls or after treating the patients. Hybridization of the PCR products with known species-specific probes of the 18S rRNA of various malaria species revealed strong positive reactions against the Plasmodium vivax-specific probe (absorbance 1.30-1.90 at 405 nm) in all of the patients. The absorbance was positively correlated with the degree of blood parasitemia, but with a borderline significance. Sequencing of the probe region of the Korean P. vivax revealed no significant variations from the typical P. vivax. The results show that the PCR-MPH is a highly useful technique for the DNA diagnosis of Korean vivax malaria.

Potentiation of the antitumor activity of 5-trifluoromethyl-2'-deoxyuridine by the use of depot forms of the parent compound.

5-Trifluoromethyl-2'-deoxyuridine (CF3dUrd), an antitumor agent, is known to be short-lived in human plasma. Since its rapid elimination from the bloodstream seems to have descouraged the clinical evaluation of this drug, we explored the potential use of masked derivatives of CF3dUrd as "depot" forms of the parent compound. First, we observed that the toxicity of CF3dUrd against HeLA cells in culture was 10(4) times greater for a 24-h treatment as compared with a 1-h treatment at identical concentrations of the drug, which suggests the importance of using a prolonged treatment period. In fact, the divided dosing of CF3dUrd to L1210-bearing mice was markedly more effective than its single administration. 5'-O-Hexanoyl-, N3-p-butylbenzoyl-, 5'-O-benzyloxy-methyl-, and 3'-O-benzyl-CF3dUrd were found to be effective in maintaining the CF3dUrd concentration in plasma. The oral doses of these agents required to achieve 50% growth inhibition (ED50) in mice bearing sarcoma 180 tumors were 19, 34, 10, and 13 mg kg-1 day-1, respectively, whereas that of CF3dUrd was 63 mg kg-1 day-1. The ED50 values for these compounds were inversely correlated with the residence time of CF3dUrd in plasma. The therapeutic indices of these compounds, calculated as the dose producing a 50% inhibition of body-weight gain (IB50) divided by the ED50 value (1.89, 1.21, 1.40, and 2.15, respectively), were significantly higher than that of CF3dUrd (0.78). Consequently, these depot forms of CF3dUrd, particularly 3'-O-benzyl-CF3dUrd, are expected to be more useful than the parent compound as antitumor agents.

Antitumor activity of FTC-092, a masked 5-trifluoromethyl-2'-deoxyuridine derivative.

1-(3-O-Benzyl-2-deoxy-beta-D-ribofuranosyl)-5-trifluoromethyl-2,4(1H,3)- pyrimidinedione (FTC-092), a fluorinated pyrimidine derivative, appeared to be effective against various transplantable tumors in mice following oral administration, and its activity was superior to that of several other antitumor fluorinated pyrimidines. The ED50 value for FTC-092 the dose effective in achieving 50% inhibition of tumor growth against the solid form of sarcoma 180 was 13.3 mg/kg daily, whereas those for 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd), the parent compound of FTC-092, for 1-(2-tetrahydrofuryl)-5-fluorouracil (Tegafur, FT), the prodrug of 5-fluorouracil (FUra), and for FUra were 64.1, 122, and 28 mg/kg daily, respectively. The therapeutic indices (LD10/ED50) of FTC-092, CF3dUrd, FT, and FUra were 4.39, 1.7, 1.35, and 1.65, respectively. FTC-092 itself is not an active agent. After it has been absorbed from the gastrointestinal tract, FTC-092 undergoes a gradual biotransformation, mainly via the action of liver microsomes, releasing CF3dUrd over a long period. The levels of CF3dUrd in the stomach and small intestine of mice after the oral administration of FTC-092 were undetectable, whereas those following the administration of CF3dUrd at the same dose were high for a period of several hours. In contrast, the CF3dUrd level generated in plasma after the administration of FTC-092 remained at a high level for a longer period than did that observed on the administration of CF3dUrd. The low levels of CF3dUrd measured in stomach and small-intestine tissues and the maintenance of CF3dUrd in blood over long periods after the administration of FTC-092 are features that favor the possible clinical application of FTC-092.

Antitumor mechanisms and metabolism of the novel antitumor nucleoside analogues, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil.

The antitumor ribonucleoside analogues 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd), first synthesized in 1995, have strong antitumor activity against human cancer xenografts without severe side effects. Here, we studied the antitumor mechanisms of ECyd and EUrd using mouse mammary tumor FM3A cells in vitro and the mechanism of selective cytotoxicity of ECyd using human tumor xenografts in nude rats in vivo. In FM3A cells, ECyd and EUrd were rapidly phosphorylated to ECyd 5'-triphosphate (ECTP) and EUrd 5'-triphosphate (EUTP), which strongly inhibiting RNA synthesis. Cells treated with EUrd were later found to contain both EUTP and ECTP, and ECTP accumulated as the final product. Probably the uracil moieties of EUrd derivatives were efficiently converted to cytosine moieties in the cells. EUrd and its derivatives were minor metabolites in the cells treated with ECyd, so cytidine forms probably were not converted to uridine forms at the nucleoside or nucleotide stage. The ultimate metabolite of ECyd and EUrd, ECTP, is stable in cultured cells with a half-life of at least 3 days, so ECyd and EUrd are on a "closed" metabolic pathway to ECTP. These characteristics of ECyd and EUrd may be important for their antitumor activity. ECyd had strong and selective antitumor activity against the human tumor xenografts. ECyd-phosphorylating activity (uridine/cytidine kinase) in the xenografts was higher than that in the organs of the rats. This finding may account for the strong activity with mild side effects. ECyd and EUrd may be a new kind of antitumor nucleoside analogue for clinical use.

Cloning and expression of uridine/cytidine kinase cDNA from human fibrosarcoma cells.

Uridine/cytidine kinase which converts uridine and cytidine to their corresponding monophosphates is a rate-limiting enzyme involved in the salvage pathway of pyrimidine synthesis. We isolated cDNA encoding the enzyme from human fibrosarcoma cells, then determined its nucleotide sequence by the 5'-RACE method followed by confirmation employing the human genome DNA library. The isolated uridine/cytidine kinase cDNA (UCK cDNA) consisted of 786 nucleotides encoding 261 amino acids and was found to have approximately 70% homology with mouse UCK cDNA. Northern blot analysis of human leukemia RNAs with labeled UCK gene showed a single band at 1.6 kb to be UCK mRNA, and southern blot analysis of the UCK cDNA after digestion with BamHI, SacI and XbaI enzymes showed four band signals, suggesting the UCK gene to have at least 4 exons. A truncated form of UCK cDNA was expressed as the His-tag conjugated protein in Escherichia coli. The expressed and purified protein specifically converted uridine and cytidine to their corresponding monophosphates and also phosphorylated antitumor nucleosides such as 5-fluorouridine, cyclopentenyl-cytosine and 3'-C-ethynylcytidine. The present results suggest that our cloned human UCK cDNA encodes the correct amino acid sequence for UCK protein, showing high intracellular phosphorylation activity forward natural and synthetic pyrimidine nucleosides.

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells.

Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2'-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.

Fecal mutagenicity arising from ingestion of fried ground beef in the human.

Fried ground beef has been shown to contain mutagens, and the major mutagenic component has been identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Mutagens in feces of 3 adult volunteers were fractionated by treatment of the feces with blue cotton followed by chromatography on a carboxymethyl cellulose column. The chromatographic fraction, corresponding to MeIQx in terms of the position of elution, was examined for mutagenicity in S. typhimurium TA98 with metabolic activation. When meals containing no heated meat were eaten, this fraction of feces showed little or no mutagenicity. On eating fried ground beef, the feces excreted in the next two days showed greatly increased mutagenicity in this fraction. By eating no-meat meal subsequent to the meat meal, the mutagenicity resumed the original low level on the fourth day after the meat meal. The components in the mutagenic fraction were probably metabolites of the mutagens present in cooked meat, since analysis by high pressure liquid chromatography of the mutagenic fraction showed that the active components in the feces were different from the mutagens in cooked meat.