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Background/aim The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). Materials and methods DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. Results IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). Conclusion IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.

Free radicals, such as metabolic intermediates, reactive oxygen species, and metal enzymes, are key substances in organisms, although they can also cause various oxidative diseases. Thus, in vivo free radical imaging should be considered as the ultimate form of metabolic imaging. Unfortunately, electron spin resonance (ESR) imaging has inherent disadvantages, such as free radicals with large linewidths generating blurred images and the presence of two or more free radicals resulting in a complicated imaging procedure. Dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) is a noninvasive imaging method to visualize in vivo free radicals, theoretically, with the same resolution as the MRI anatomical resolution, and fixed low-field DNP-MRI provides unique information on oxidative diseases and cancer. However, the large gyromagnetic ratio of the electron spin, which is 660-fold greater than that of a proton, requires field cycling, wherein the external magnetic field should be varied during DNP-MRI observations. This causes difficulties in developing a DNP-MRI system for clinical purposes. We developed a novel field-cycling DNP-MRI system for a preclinical study. In the said system, the magnetic field is switched by rotationally moving two magnets, with a magnetic flux density of 0.3 T for MRI and 5 mT for ESR. The image quality was examined using various pulse sequences and ESR irradiation using nitroxyl radical as the phantom, and the optimum conditions were established. Using the system, we performed a preclinical study involving free radical imaging by placing the free radicals under the palm of a human hand.

DNA has been recognized as an ideal material for bottom-up construction of nanometer scale structures by self-assembly. The generation of sequences optimized for unique self-assembly (GENESUS) program reported here is a straightforward method for generating sets of strand sequences optimized for self-assembly of arbitrarily designed DNA nanostructures by a generate-candidates-and-choose-the-best strategy. A scalable procedure to prepare single-stranded DNA having arbitrary sequences is also presented. Strands for the assembly of various structures were designed and successfully constructed, validating both the program and the procedure.

Probe-free imaging of redox intermediate radicals must become a crucial method and diagnosis. DNP (dynamic nuclear polarization)-MRI, a new imaging method for free radical (Lurie, et al. 1988), utilizes Overhauser effect that nuclear spin close to free radical is 300 times higher polarized than that of natural state by inducing the electron spin resonance of free radical. The advantage of DNP-MRI is the high sensitivity and resolution, theoretically similar to that in MRI. We installed the custom-made DNP-MRI from Philips and succeeded in the imaging of redox status in disease models and the spectroscopic imaging of redox intermediate radicals from FMN, FAD, CoQ10, and vitamin E. It has, however, the large disadvantage of poor sensitivity and heating by ESR irradiation, because of its low magnetic field (15mT MRI). Here, we developed the novel field-cycling DNP-MRI system for clinical trial, in which free radicals are visualized with 0.3T MRI as the polarized proton by irradiating the free radicals at 5mT, and demonstrated the clear images of the phantoms containing FAD radical with less than 0.5 mm of the spatial resolution. The image of FAD radical placed under healthy volunteer hand was superimposed on the anatomical MRI image. The intrinsic radical, melanin, could be also visualized. Overhauser effect is known to be occurred in the various kind of free radicals, metal-complex, etc., and also in lipid membranes. The newly developed DNP-MRI could demonstrate the functional/metabolic imaging by visualizing intrinsic radials, redox intermediates and/or metal enzymes in disease models and also in patients.

Instability of short tandem repeats (STRs) in DNA during replication is observed in all organisms examined, and is causatively involved in various human diseases. We explore the mechanisms involved in instability by examining length changes occurring during the replication of [(CA) 20 TA] n and [(CAG) 20 TAG] n , in human cells. We show that the majority of alterations consist of an insertion or deletion of one repeat unit, and base substitutions or length changes involving many repeat units are rare. We also show that length changes of two-tract STRs are biased toward the 3′-end of the repeat tract, in reference to lagging strand synthesis. There are some differences between our observations and previous observations in microbes, e.g. the orientation effect was not observed in this study. The results of this study are discussed in terms of the molecular mechanisms leading to alterations in repeat tracts.

Effective use of knowledge of human genome sequences in studies of hereditary diseases or cancer heavily depends on efficient methods for detection of mutations in individual samples. We describe here a simple and efficient mutation scanning system in which PCR products arepost-labeled with two different fluorescent dyes in one tube, and analyzed by an automatedcapillary electrophoresis system using single-strand conformation polymorphism (SSCP) conditions (PLACE–SSCP). With the appropriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, then the presence of mutations is statistically judged. Thirty-three of 34 known mutations in fragments of three unrelated sequence contexts up to 741 bp were detected using one electrophoresis condition at the confidence level of

Sensitivity of single-strand conformation polymorphism analysis of PCR products (PCR-SSCP analysis) is known to be decreased when the DNA fragments are longer than 300 bp. We examined effects of buffer ions in an attempt to extend the length limit of the analysis. It has been noted that addition of glycerol to the gel containing Tris-borate buffer enhances the sensitivity, but the effects of glycerol have been left unexplained. We found that the effects of glycerol are caused by the reduction of pH of the buffer by the reaction of glycerol and borate ion. We further extended these observations and found that sensitivity of SSCP can be greatly improved by running the electrophoresis in low pH buffer systems. Using a new buffer system and running the electrophoresis at a fixed temperature, we detected 27 of 31 known mutations of factor IX gene in six different sequence contexts ranging in length from 300 to 800 bp.

The single strand conformation polymorphism (SSCP) method is a sensitive technique used to detect subtle sequence differences in PCR-amplified DNA fragments as separated peaks in electrophoretic analysis. In this chapter, we focus on SSCP analysis for quantifying polymorphic alleles rather than scanning for mutations. Short fragments carrying single nucleotide polymorphisms are amplified from individual and pooled DNA samples, then the products are labeled with fluorescent dyes and analyzed by automated capillary electrophoresis under nondenaturing conditions. Dedicated software, QSNPlite, interprets trace data of the electrophoresis to identify alleles of individuals and quantify these alleles in the pool. The software can also incorporate sequencing data to assign alleles at the nucleotide level. The procedures described here are being used in association studies that compare allele frequencies between cases and controls to identify genes responsible for common diseases.

A method for fluorescent postlabeling of PCR products has been developed. The method uses Klenow fragment of DNA polymerase I that exchanges the 3'-terminal residue of PCR-amplified DNA fragment for fluorescent nucleotides. All reactions, including PCR, are performed in one tube simply by successive addition of reagents. The products can be applied directly to fluorescence-based automated DNA sequencers without purification for either length determination in denaturing electrophoresis or mutation detection in SSCP electrophoresis.

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.

Thioproline, which is readily nitrosated to form nitrosothioproline, is expected to act as a nitrite scavenger. The effect of thioproline as an inhibitor of the carcinogenesis induced by N-nitroso-N-benzylmethylamine precursors was examined. Two groups of male F-344 rats were given diet containing 0.25% N-benzylmethylamine (group I) or 0.25% N-benzylmethylamine plus thioproline (0.25% until wk 17 and then 0.5%; group II). Both groups were given drinking-water containing sodium nitrite (0.1% until wk 17 and then 0.2%). The experiment was continued for 717 days. Squamous cell carcinoma of the forestomach developed in six out of seven rats in group I and in significantly fewer, two out of nine rats, in group II. The degree of invasion by the tumours was also less in group II rats, given thioproline, than in group I. Thus thioproline suppressed carcinogenesis induced by N-benzylmethylamine and nitrite, possibly by inhibiting the in vivo nitrosation of N-benzylmethylamine.

Two indole compounds isolated from fresh Chinese cabbage were shown to be mutagen precursors that yielded direct-acting mutagens on treatment with nitrite. They were identified as 4-methoxyindole-3-acetonitrile and 4-methoxy-indole-3-aldehyde. When these compounds were treated with 50 mM nitrite at pH 3.0 for 1 h at 37 degrees C, 4-methoxyindole-3-acetonitrile induced 31,800 and 10,000 revertants of Salmonella typhimurium TA100 and TA98, respectively, per mg of mutagen precursor in the absence of S9 mix, and 4-methoxyindole-3-aldehyde induced 156,900 revertants of TA100 and 26,800 revertants of TA98 per mg in the absence of S9 mix.

After nitrite treatment, various kinds of pickled vegetables and sun-dried fishes produced in Japan showed direct-acting mutagenicity on Salmonella typhimurium TA100, inducing 1900-18000 revertants/g. Kimchis, sun-dried fishes, sun-dried squid, soy sauces, fish sauces, bean pastes and shrimp paste produced in Korea, the Philippines and Thailand also showed direct-acting mutagenicity after nitrite treatment. All soy sauces and fish sauces tested contained as much tyramine as 17-1020 micrograms/ml, but very low or undetectable amounts of (-)-(1S,3S)- and (-)-(1R,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids.

After treatment with nitrite, Chinese cabbage showed direct-acting mutagenicity on Salmonella typhimurium TA100 inducing 3100 revertants per g. One of the mutagen precursors that became mutagenic after nitrite treatment was isolated, and identified as indole-3-acetonitrile. After treatment with nitrite, 1 mg of indole-3-acetonitrile induced 17 400 revertants of TA100 and 21 000 revertants of TA98 without S9 mix.

Twelve aplysiatoxin compounds have been evaluated as possible tumor promoters in vivo by means of three biological tests: viz. irritation of mouse ear, induction of ornithine decarboxylase in dorsal skin of mice, and inhibition of specific binding of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to an epidermal particulate fraction. The potencies of these three biological activities correlate well for each derivative. Bromoaplysiatoxin shows biological activities that are similar to those of the strong tumor promoter, aplysiatoxin. The present studies suggest that the C-3, C-20 and C-30 hydroxyl groups of the aplysiatoxins are involved in binding to the specific receptor of TPA.

Soy sauce treated with nitrite was found to be more mutagenic to Escherichia coli WP2 uvrA/pKM101 than Salmonella typhimurium TA100 without S9 mix. The mutagenicity of soy sauce treated with nitrite was affected by the concentration of soy sauce in the nitrosation mixture, and a concentration of 5% resulted in the highest specific activity (revertants/ml soy sauce equivalent). By incubating soy sauce at a concentration of 5% in a solution of 1 mM nitrite at pH 3 for 1 h at 37 degrees C, the equivalent of 1 ml of soy sauce induced 2790 revertants of E. coli WP2 uvrA/pKM101 without S9 mix.

Oncogenes of fibrosarcomas of rats, induced by subcutaneous injection of 1,8-dinitropyrene (1,8-DNP), were examined by NIH 3T3 cell transfection assay and Southern blot analysis. Transformants containing rat specific repetitive sequences were obtained with DNAs of 4 fibrosarcomas, 1,8-DNP1, 1,8-DNP2, 1,8-DNP3 and 1,8-DNP7. A transformant, 1,8-DNP2-2, induced by DNA of a fibrosarcoma, 1,8-DNP2, and 7 secondary transformants derived from it contained rat K-ras sequences. Another transformant, 1,8-DNP2-1, induced by the same sarcoma did not have a ras family oncogene. This indicates that the sarcoma, 1,8-DNP2, has at least 2 transforming genes. The transforming genes of 6 other transformants derived from 3 sarcomas did not contain ras family or neu transforming genes.

Thapsigargin, a hexaoxygenated tetraacylated sesquiterpene lactone, induced irritation of mouse ear and histidine decarboxylase (HDC) activity in mouse skin, but it did not induce ornithine decarboxylase in mouse skin or adhesion of human promyelocytic leukemia (HL-60) cells. Although thapsigargin did not give consistent positive results in a short-term screening system for tumor promoters, it was tested in a two-stage carcinogenesis experiment on mouse skin. The potency of thapsigargin to induce HDC in mouse skin was used to determine the dose in this experiment. Application of 10 micrograms (17 nmol) thapsigargin induced HDC activity of 139 pmol CO2/mg protein per 60 min. Tumors were found in the skin of 53.5% of the mice treated with DMBA plus 5 micrograms (8.5 nmol) thapsigargin in week 22, in none of those treated with thapsigargin alone by week 30. One tumor appeared in 1 of 15 mice treated with DMBA alone in week 21. Thapsigargin cannot bind to the phorbol ester receptor in the particulate fraction of mouse skin and so is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA) type tumor promoter. It is a new tumor promoter differing in many respects from the well-defined TPA type tumor promoters. Several naturally occurring analogues of thapsigargin, such as thapsigargicin and thapsitranstagin, might also be new non-TPA type tumor promoters, because thapsigargicin and thapsitranstagin induced irritation of mouse ear and HDC activity in mouse skin.

Broiled chicken, pork, mutton, beef and sun-dried sardine were found to yield direct-acting mutagenicity after nitrite treatment. When 50% methanol extracts of cooked foods were treated with 50 mM nitrite at pH 3 for 1 h at 37 degrees C, they induced 3800-17,900 revertants of Salmonella typhimurium TA100 and 15,000-43,600 revertants of TA98 per g. In contrast, raw meat and uncooked sun-dried sardine showed little or no mutagenicity after nitrite treatment. Treatment of broiled chicken with 0.5-3 mM nitrite, which is a physiologically feasible concentration in the human stomach under some conditions, induced direct-acting mutagenicity. When broiled chicken was treated with 1 mM nitrite at pH 3 for 1 h at 37 degrees C, its mutagenicities on TA100 and TA98 without S9 mix were 7100 and 5400 revertants/g, respectively.