Antimalarial activity of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) and its carboxylic acid derivatives.

Malaria is one of the world's deadliest diseases and is becoming an increasingly serious problem as malaria parasites develop resistance to most of the antimalarial drugs used today. We previously reported the in vitro and in vivo antimalarial potencies of 1,2,6,7-tetraoxaspiro[7.11]nonadecane (N-89) and 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Plasmodium falciparum and Plasmodium berghei parasites. To improve water-solubility for synthetic peroxides, a variety of cyclic peroxides having carboxyl functionality was prepared based on the antimalarial candidate, N-251, and their antimalarial activities were determined. The reactions of N-89 and its derivatives with Fe(II) demonstrated a highly efficient formation of the corresponding carbon radical which may be suspected as a key for the antiparasitic activity.

Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol.

Plasmodium falciparum, the major causative parasite for the disease, has acquired resistance to most of the antimalarial drugs used today, presenting an immediate need for new antimalarial drugs. Here, we report the in vitro and in vivo antimalarial activities of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against P. falciparum and Plasmodium berghei parasites. The N-251 showed high antimalarial potencies both in the in vitro and the in vivo tests (EC(50) 2.3×10(-8) M; ED(50) 15 mg/kg (per oral)). The potencies were similar to that of artemisinin in vitro and greater than artemisinin's activity in vivo (p.o.). In addition, N-251 has little toxicity: a single oral administration at 2000 mg/kg to a rat gave no health problems to it. Administration of N-251 to mice bearing 1% of parasitemia (per oral 68 mg/kg, 3 times a day for 3 consecutive days) resulted in a dramatic decrease in the parasitemia: all the 5 mice given N-251 were cured without any recurrence, with no diarrhea or weight loss occurring in the 60 days of experiment. N-251 deserves more extensive clinical evaluation, desirably including future trials in the human.

Progress in the bisulfite modification of nucleic acids.

Bisulfite modification is a principal tool for analyzing DNA methylation, the methyl substitution at position 5 of cytosine residues. Hypermethylation is known to cause silencing of genes, which may result in cell function failures. DNA methylation analysis is therefore a focus of attention in various fields of biological sciences, including even clinical practices for treatment of cancer patients. In 2004, we reported that the bisulfite modification of DNA necessary in this analysis can be speeded up significantly by using a high concentration ammonium bisulfite solution (10 M), in place of traditional sodium bisulfite solution of 5 M concentration. Evaluations on this newer protocol have now come out from several laboratories, showing that this quick process can yield results with greater accuracy compared to those obtainable with widely-practiced low-concentration methods. Another aspect reported here is a study on the desulfonation of uracil-bisulfite adduct to form uracil, the last step of the bisulfite-conversion of cytosine to uracil. Kinetic measurements for the desulfonation of uridine-bisulfite adduct at a near-neutral pH region are described.

Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account.

Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.

X-ray crystallographic identification of bisulfite-uracil adduct as sodium 5,6-dihydrouracil 6-sulfonate.

DNA methylation at position 5 of cytosine residues plays an important role in the gene function control. The analytical method for determining the sites of 5- methylcytosine residues utilizes bisulfite treatment of genomes. Cytosines in DNA are converted into uracils by this treatment, while 5-methylcytosines remain unaltered. The bisulfite treatment followed by amplification by polymerase chain reaction and by sequencing the resulting DNA allows determination of the 5-methylcytosine sites in the original. In this chemical modification, key intermediates are those formed by addition of bisulfite across the 5,6-double bond of pyrimidine ring. Their structures were proposed in 1970 as 5,6-dihydropyrimidine 6-sulfonates, but not its 6-sulfurous acid ester, on the basis of spectral data. X-ray analysis has now been performed for a single crystal of sodium bisulfite-uracil adduct and the results showed its structure as sodium 5,6-dihydrouracil 6-sulfonate monohydrate, thus providing definite evidence for the C(6)-sulfonate structure.

Bisulfite modification for analysis of DNA methylation.

Bisulfite is known to deaminate cytosine in nucleic acids, while 5-methylcytosine resists this bisulfite action. For this reason, bisulfite treatment has been used for detecting 5-methylcytosine in DNA, a minor component of eukaryotic DNA, presently recognized as playing an important role in the control of gene function. This procedure, called bisulfite genomic sequencing, is a principal method for the analysis of DNA methylation in various biological phenomena, including human diseases such as cancer. This unit describes an efficient procedure utilizing a newly developed high-concentration bisulfite solution. Protocols for this methodology are supplemented with discussions focused on chemical aspects of the bisulfite treatment.

The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research.

Methylation at position 5 of cytosine in DNA is an important event in epigenetic changes of cells, the methylation being linked to the control of gene functions. The DNA methylation can be analyzed by bisulfite genomic sequencing, and a large body of data have now been accumulated, based on which causation of diseases, for example cancer, and many other manifestations of cellular activities have been discussed intensively. This article gives an extensive account of the chemical aspects of bisulfite modification of cytosine and 5-methylcytosine in DNA. Various factors that affect the action of bisulfite are discussed, and a recent progress from our laboratory is explained. Conventional procedures for the bisulfite treatment consist incubation of single-stranded DNA with sodium bisulfite under acidic conditions. This treatment converts cytosine into uracil, but 5-methylcytosine remains unchanged. Amplification by polymerase chain reaction (PCR) of the bisulfite-treated DNA followed by sequencing can result in revealing the positions of 5-methylcytosine in the gene. We have discovered that the whole procedure can be significantly speeded up by the use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. Another recent finding is that urea, which has been often added to the reaction mixture with the purpose of facilitating the bisulfite-mediated deamination of cytosine in DNA, may not work as anticipated: we have observed that urea does not show such promoting actions in our treatments of DNA. A laboratory protocol for quantifying bisulfite, suitably simple for routine practice to ensure valid experiments, is described.

Chemistry of bisulfite genomic sequencing; advances and issues.

Methylation at position 5 of cytosine in DNA plays a major role in epigenetic gene control. The methylation analysis can be performed by bisulfite genomic sequencing. Conventional procedures in this analysis include a treatment of single stranded DNA with 3-5 M sodium bisulfite at pH 5 and at 50-55 degrees for 4-20 hr. This will convert cytosine into uracil, while 5-methylcytosine resists this deamination. Amplification by PCR of the bisulfite-treated DNA followed by sequencing reveals the positions of 5-methylcytosine in the gene. We reported recently that the whole procedure can be speeded up by use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. We also reported that urea, which has been often added to the reaction mixture with the purpose of facilitating the reaction, may not work as anticipated. This time, we would like to address the need for further investigating the chemistry of the bisulfite modification of DNA. Particularly important is to study side reactions that may occur due to the exhaustive bisulfite treatment required for achieving complete deamination of all the cytosine residues in a given sample of DNA.

Does urea promote the bisulfite-mediated deamination of cytosine in DNA? Investigation aiming at speeding-up the procedure for DNA methylation analysis.

Methylation of cytosine in DNA at position 5 plays important roles in gene functions. Changes in the methylation status are linked to cancer. These studies have been developed on the basis of determining 5-methylcytosine residues [mC] in DNA. This analytical procedure uses the principle that bisulfite deaminates cytosine [C] but it deaminates mC only very slowly. Thus, 'bisulfite genomic sequencing' involves treatment of a given DNA sample with bisulfite followed by PCR amplification and sequencing, through which C residues in the original DNA are found as T and mC as C. In this procedure, a treatment with 3-5 M sodium bisulfite for 12-16 hr at 55 degrees C has been conventionally used. Recently, we were able to improve the efficiency of this procedure by introducing a highly concentrated (10 M) bisulfite solution. Aiming at further improvement of the procedure, we have now explored the effect of adding urea in this bisulfite treatment, as urea was reported to improve the deamination efficiency. Using 7.5 M ammonium bisulfite (pH 5.4) at 70 degrees C with or without the presence of 6 M urea, we performed deamination and sequencing of a DNA sample having known multiple CpG sites with mC. The deaminated DNAs were then subjected to PCR amplification followed by sequencing. In the 15 min-treated sample, the deamination extents were; C 96.5%, mC 1.1% for "bisulfite-only"; and C 90.3%, mC 1.4% for "bisulfite + urea". In the 30 min-treated sample, these values were; C 99.7%, mC 3.6% for "bisulfite only"; and C 99.7%, mC 2.1% for "bisulfite + urea". These results indicate that urea did not enhance the deamination efficiency. In the PCR, we did not observe significant improvements regarding the amounts of DNA necessary to obtain adequate amplification. Urea at 2 M, 4 M, and 8 M, showed no improvements. We conclude that urea gave no significant effect in the bisulfite genomic sequencing of the DNA used.

Characterization of adducts formed in the reaction of 2-chloro-4-methylthiobutanoic acid with 2'-deoxyguanosine.

2-chloro-4-methylthiobutanoic acid (CMBA) is a direct-acting mutagen found in salt-nitrite-treated Sanma fish or similarly treated methionine solution. In this study, CMBA was reacted with 2'-deoxyguanosine (dG) in phosphate buffer (pH 7.4) at 37 degrees C. The HPLC-UV analysis showed that two products were mainly formed during the reaction. These were isolated, purified by semipreparative HPLC, and characterized as N7-guanine adducts: N7-(3-carboxy-3-methylthiopropyl)guanine (A1) and N7-(1-carboxy-3-methylthiopropyl)guanine (A2). Furthermore, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis was employed to investigate the possible formation of minor products during the time-course of the reaction of CMBA with dG. It was found that N7-dG adducts, the precursors of A1 and A2, were formed early in the reaction and that subsequently the spontaneous depurination occurred to yield stable N7-guanine adducts A1 and A2. Stability studies in phosphate buffer (pH 7.4) at 37 degrees C showed that the amount of each N7-dG adduct decreased rapidly with a half-life of 6 h and 4 h to yield A1/A2, respectively. A regioisomer of N7-dG adducts was also observed in the LC/ESI-MS/MS analysis, but it was not characterized in detail because it was present only in trace amounts. On the basis of structural features, A1 and A2 seemed to be formed from the reaction of dG with 1-methyl-2-thietaniumcarboxylic acid, an intermediate resulting from the cyclization of CMBA. However, A2 might also have formed from the direct reaction of dG and CMBA. N7-Alkylation of the guanine residue and subsequent depurination are known to produce apurinic sites in DNA that induce point mutations and may be responsible for the observed CMBA-induced mutagenesis.

Effect of chlorophyllin-chitosan on excretion of dioxins in a healthy man.

We investigated the usefulness of chitosan and chlorophyllin-chitosan (chl-chitosan) administration for reduction of the body burden of environmental dioxins, including polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/ Fs) and coplanar polychlorinated biphenyls (Co-PCBs), by examining the excretion levels in the feces and sebum of a healthy man. The volunteer ate the same three meals every day during the 40-d experiment, which was composed of five phases (I-V) of 8 d each. In phase I (days 1-8), the volunteer was given only the basal diet. In phases II-V, 0.2 g of chitosan, 0.6 g of chitosan, 0.2 g of chl-chitosan, and 0.6 g of chl-chitosan, respectively, were administered immediately after each meal. We measured daily the amount of dioxins occurring in the feces and sebum during the last 5 d of each phase. The total toxicity equivalency (TEQ) of the dioxin in phases I-V were 27, 26, 38, 36, and 67 pg/d in the feces and 20, 19, 16, 16, and 14 pg/d in the sebum, compared with 74 pg/d in the food. The excretion of dioxins in the feces was significantly increased in phases III, IV, and V, being 140% (p < 0.05), 135% (p < 0.05), and 249% (p < 0.01) of the control level (phase I). Although the dioxin in the sebum was slightly decreased in phase V as compared with the control level, the total amount of excreted dioxin in feces and sebum was increased significantly in phase V, being 174% of the control level, which is almost the same level as that in the food. This indicates that chl-chitosan can prevent accumulation of dioxin, at least at the intake level of normal foods.

No effects of chlorophyllin on IQ (2-amino-3-methylimidazo[4,5-f]-quinoline)-genotoxicity and -DNA adduct formation in Drosophila.

Previously we demonstrated that chlorophyllin suppressed the genotoxicities of many carcinogens. However, the genotoxicity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline), a carcinogenic heterocyclic amine, was not suppressed in Drosophila. On the contrary, it has been reported that chrolophyllin suppressed the genotoxicity of IQ in rodents, rainbow trout and Salmonella. We demonstrated that the chlorophyllin-induced suppression of MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline)-genotoxicity was associated with a decrease in MeIQx-DNA adduct formation in Drosophila larval DNA. MeIQx represents another type of heterocyclic amine and is similar to IQ in structure. In this study we utilized (32)P-postlabeling to examine whether chlorophyllin reduced IQ-DNA adduct formation in Drosophila DNA in the same way as MeIQx. The results revealed that the formation of IQ-DNA adducts was unaffected by treatment with chlorophyllin. This was consistent with the absence of any inhibitory effect on genotoxicity as observed in the Drosophila repair test. These results suggest that IQ-behavior in Drosophila is not affected by chlorophyllin, indicating that the process of IQ-DNA adduct formation followed by expression of genotoxicity in Drosophila may be different from that in other organisms.

Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis.

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The determination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 degrees C. We demonstrate here that this deamination can be speeded up significantly by increasing the bisulfite concentration and the temperature with which the reaction is performed. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at pH 5.4 and 90 degrees C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer an improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.

High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation.

Bisulfite genomic sequencing is a widely used technique for analyzing cytosine-methylation of DNA. By treating DNA with bisulfite, cytosine residues are deaminated to uracil, while leaving 5-methylcytosine largely intact. Subsequent PCR and nucleotide sequence analysis permit unequivocal determination of the methylation status at cytosine residues. A major caveat associated with the currently practiced procedure is that it takes 16-20 hr for completion of the conversion of cytosine to uracil. Here we report that a complete deamination of cytosine to uracil can be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature. Time course experiments demonstrated that treating DNA with 9 M bisulfite for 20 min at 90 degrees C or 40 min at 70 degrees C all cytosine residues in the DNA were converted to uracil. Under these conditions, the majority of 5-methylcytosines remained intact. When a high molecular weight DNA derived from a cell line (containing a number of genes whose methylation status was known) was treated with bisulfite under the above conditions and amplified and sequenced, the results obtained were consistent with those reported in the literature. Although some degradation of DNA occurred during this process, the amount of treated DNA required for the amplification was nearly equal to that required for the conventional bisulfite genomic sequencing procedure. The increased speed of DNA methylation analysis with this novel procedure is expected to advance various aspects of DNA sciences.

Inhibitory effects of (-)-epigallocatechin gallate on the mutation, DNA strand cleavage, and DNA adduct formation by heterocyclic amines.

Green tea is known to be a potential chemopreventive agent against cancer. In this study, we investigated the inhibitory activities of tea extracts, and in particular the polyphenolic component (-)-epigallocatechin gallate (EGCG), against heterocyclic amine-induced genotoxicity. The tea extracts displayed inhibition of 2-hydroxyamino-6-methyldipyrido[1,2-a,3',2'-d]imidazole (Glu-P-1(NHOH))-induced mutagenicity. This inhibition can be accounted for by the presence of EGCG in the extracts. The mutagenic effect of Glu-P-1(NHOH), which induces single-strand cleavage in supercoiled circular DNA under neutral conditions, was inhibited by EGCG. Using the Drosophila repair test, a test for gross DNA damage, and DNA adduct detection by (32)P-postlabeling, we showed that EGCG prevented 2-amino-3,8-dimethylimidazo[4,5-f]quinoline-induced DNA damage and adduct formation in insect DNA. EGCG was found to accelerate the degradation of Glu-P-1(NHOH) in vitro. This observation suggested that the inhibition by EGCG is associated with an accelerated degradation of metabolically activated heterocyclic amines.

Protection against Trp-P-2 DNA adduct formation in C57bl6 mice by purpurin is accompanied by induction of cytochrome P450.

Purpurin, an anthraquinone constituent from madder root, has previously been reported as antimutagenic in the Ames Salmonella bacterial mutagenicity assay and as antigenotoxic in Drosophila melanogaster, against a range of environmental carcinogens. Short-term dietary supplementation with purpurin inhibits the formation of hepatic DNA adducts in male C57bl6 mice after a single dose of the heterocyclic amine dietary carcinogen Trp-P-2 (30 mg/kg). Inhibition of adduct formation was dose-dependent. No DNA adducts were observed in animals treated only with purpurin. The decrease in adduct formation was accompanied by significant, dose-dependent inductions of hepatic cytochrome P450-dependent dealkylations of methoxy- (CYP1A2), ethoxy- (CYP1A1), and pentoxy- (CYP2B) resorufins, total cytochrome P450, and NADPH cytochrome P450 reductase. It is hypothesized that purpurin exhibits chemopreventive potential by inhibiting the cytochrome P450-dependent metabolism of heterocyclic amines to their genotoxic N-hydroxylamines.

Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity.

N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite. NPRO has been reported to be nonmutagenic and noncarcinogenic. In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium. Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight. The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA. Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay. An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks. The formation of nitric oxide was observed in NPRO solution irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm. Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm. The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation.

Preventive effects of chlorophyllin fixed on chitosan towards DNA adduct formation of 3-amino-1-methyl-5H-pyrido [4,3-b]indole in CDF1 mice.

Chlorophyllin, a water-soluble derivative of chlorophyll, is known to suppress the mutagenic and carcinogenic action of compounds having polycyclic structures, e.g., heterocyclic amines and aflatoxin B1. Recently, we reported that chlorophyllin fixed on chitosan (chl-chitosan), which is insoluble in water, can efficiently and tightly trap these heterocyclic amines. We have studied whether this adsorption to chl-chitosan can result in an interference with DNA-adduct formation caused by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a heterocyclic amine, in CDF1 mice, in which Trp-P-2 had been shown to induce hepatocellular carcinomas. Mice were fed a diet containing Trp-P-2 with or without chl-chitosan. After 3 d of feeding, DNA-adduct formation in liver and lung was examined by 32P-postlabeling analysis. Adducts formed from Trp-P-2 were significantly decreased by the chl-chitosan addition (p<0.05, t-test). These results suggest that the uptake of Trp-P-2 into the mouse was lowered by its adsorption to chl-chitosan, either within the digestive tract or within the food itself. This trapping agent, chl-chitosan, is thus worthy of study for cancer chemoprevention.