Katsutoshi Ohno

A genotoxicity test system based on p53R2 gene expression in human cells: Assessment of its reactivity to various classes of genotoxic chemicals

The tumor suppressor, p53, plays an important role in DNA damage repair, by regulating the expression of target genes. One p53-target gene, p53R2, which encodes a subunit of ribonucleotide reductase, is activated by DNA damage. We have previously developed a genotoxicity test system, using human cell lines and a p53R2-dependent luciferase reporter gene assay. 80 chemicals have been examined with this system and 40 of 43 Ames-positive chemicals induced luciferase activity. Eight Ames-negative genotoxic chemicals also induced luciferase activity. Although this assay system could, potentially, be applied to the rapid screening of chemicals that are potentially genotoxic to humans, the ability of the assay to detect genotoxic effects was unclear. In this study, to evaluate the performance of this assay system, several different types of DNA damaging agents were screened. 27 chemicals, whose genotoxic mechanisms are well known, were screened. All genotoxic compounds, except for anti-metabolites and histone deacetylase HDAC inhibitors, showed significant luciferase activity with the following rank order of potency: topoisomerase II inhibitors, intercalaters>bleomycin>topoisomerase I inhibitors>alkylating agents=DNA cross-linking agents=polycyclic aromatic hydrocarbons>spindle poisons. This assay showed greater response to those genotoxic agents that induce DNA double strand break damage compared to those agents that cause other forms of DNA damage. DNA double strand breakage initiates genomic instability, a feature of carcinogenicity. These results indicate that this assay system could be a helpful tool for predicting chemical genotoxicity and carcinogenicity in humans.

A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus.

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.

Validation of a genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells.

Genotoxicity assessment is important for predicting the carcinogenicity of chemical substances. p53R2 is a p53-regulated gene that is induced by various genotoxic stresses. We previously developed a p53R2-dependent luciferase reporter gene assay in the MCF-7 human breast adenocarcinoma cell line, and demonstrated its ability to detect genotoxic agents. In this paper, we investigate the applicability of the p53R2-based genotoxicity test in the human lymphoblastoid cell line TK6. TK6 cells that express wild-type p53 have been widely used for genetic toxicology studies. To evaluate the performance of the test system in TK6 cells, we referred to 61 of the chemicals on the list of 20 genotoxic and 42 non-genotoxic chemicals recommended for the evaluation of modified or new mammalian cell genotoxicity tests by the European Centre for the Validation of Alternative Methods. The overall accordance, sensitivity, and specificity of our results with the ECVAM list were 90% (55/61), 85% (17/20), and 93% (38/41), respectively. These results indicate that the p53R2-based genotoxicity test can detect various types of genotoxic chemicals without compromising its specificity. This test will be a valuable tool for rapid screen for identifying chemicals that may be genotoxic to humans.

Validation of an in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expression

Chemical carcinogenesis is a multifactorial process comprising two main stages: initiation and promotion. Tumor promoters cause the development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The identification of tumor promoters is important for preventing cancer. We previously identified 22 specific gene markers using a global gene expression analysis of chemically induced tumor promotion and established an in vitro real-time PCR screening assay for the assessment of the tumor promoting potential of chemicals in BALB/c 3T3 cells. Our in vitro tumor promoter screening test, based on these marker genes, enables earlier assessment, and is easier to conduct than classical methods. The general applicability of these markers, however, was unknown. In this study, to evaluate the performance of a set of markers, we independently validated a separate sample set, which had various structures and properties. Independent validation of the signature of 63 test chemicals showed an accuracy, sensitivity, and specificity of the assay of 96.8%, 97.0% and 96.7%, respectively. These results indicate that the tumor promoting activity assay, based on the expression of 22 marker genes, will become a valuable tool for rapid screening of potential tumor promoters.

Simultaneous detection of eight species of tree nut in foods using two tetraplex polymerase chain reaction assays

ABSTRACT Tree nuts comprise a category of food allergens that must be included in the food labels in several countries. We developed a polymerase chain reaction (PCR) method using eight specific primer pairs to detect eight representative tree nuts (almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, and walnut) under the same experimental conditions. The specificity of the eight primer pairs was confirmed by PCR testing against a variety of plant and animal samples. The detection limit of the method ranged from 1 fg to 1 pg DNA of individual tree nuts. The method detected tree nut DNA in processed and unprocessed food. In addition, the primer pairs could be combined into two sets of tetraplex PCR system. The developed method is specific, sensitive, and efficient, making it useful for detecting trace amounts of eight species of tree nut in foods. GRAPHICAL ABSTRACT Detection of eight species of tree nut (almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, and walnut) by two sets of tetraplex PCR system.