Satoko Ishikawa

A practical approach for the chemical synthesis of 2′-deoxyguanosine-C8 adducts with mutagenic/carcinogenic amino- or nitro-arenes

Abstract Synthetic methods for the preparation of 2′-deoxyguanosine-C8 (dG-C8) adducts with several mutagenic and carcinogenic amino- or nitro-arenes were developed using the palladium-mediated cross-coupling reaction of protected 8-amino-dG with bromoarenes in around 80% yields, followed by conventional deprotection procedures. This approach can be applied to preparation of a variety of authentic dG-C8 adducts with amino or nitro-arenes.

7-Azabicyclo[2.2.1]heptane as a structural motif to block mutagenicity of nitrosamines

Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications.

Antimutagenic components in Glycyrrhiza against N-methyl-N-nitrosourea in the Ames assay

Abstract Antimutagenesis against N-nitroso compounds contribute to prevention of human cancer. We have found that Glycyrrhiza aspera ethanolic extract exhibits antimutagenic activity against N-methyl-N-nitrosourea (MNU) using the Ames assay with Salmonella typhimurium TA1535. In the present study, eight purified components from Glycyrrhiza, namely glabridin, glycyrrhetinic acid, glycyrrhizin, licochalcone A, licoricesaponin H2, licoricesaponin G2, liquiritigenin and liquiritin were evaluated for their antimutagenicity against MNU in the Ames assay with S. typhimurium TA1535. Glycyrrhetinic acid, glycyrrhizin, licoricesaponin G2, licoricesaponin H2 and liquiritin did not show the antimutagenicity against MNU in S. typhimurium TA1535. Glabridin, licochalcone A and liquiritigenin reduced revertant colonies derived from MNU in S. typhimurium TA1535 without showing cytotoxic effects, indicating that these compounds possess antimutagenic activity against MNU. The inhibitory activity of glabridin and licochalcone A was more effective than that of liquiritigenin. Thus, Glycyrrhiza contains antimutagenic components against DNA alkylating, direct-acting carcinogens.

Mutagenicity of polyaromatic hydrocarbons by chemical models for cytochrome P450 in Ames assay

DNA damage is an important step in carcinogenesis. The Ames assay is a short-term screening of carcinogens that induce DNA damage. Most carcinogens require enzymatic activation through oxidation by cytochrome P450 (CYP450) in the presence of S9 mix. A combination of iron (Fe)(III) porphyrin and an oxidant is also able to oxidize compounds as an alternative metabolic pathway to CYP450. Previously it was reported that a chemical model containing a water-soluble 5,10,15,20-tetrakis(1-methylpyridinium4-yl)porphyrinatoiron(III) chloride (4-MPy) and tert-butyl hydroperoxide (t-BuOOH) activated aromatic amines and amides. In this study, a chemical model composed of an Fe porphyrin, water-insoluble 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (F5P) or water-soluble 4-MPy was optimized with an oxidant – t-BuOOH, magnesium monoperoxyphthalate (MPPT), or iodosylbenzene (PhIO). Subsequently the mutagenicity of benzo[a]pyrene (B[a]P) and chrysene in Salmonella typhimurium TA strains was compared. B[a]P was activated by a combination of F5P or 4-MPy plus MPPT or PhIO in S. typhimurium TA1538. The B[a]P-induced mutagenicity with F5P plus oxidant was higher than 4-MPy plus oxidant. Mutagenicity of chrysene, a tetracyclic aromatic hydrocarbon, was not detected in the presence of F5P/PhIO in S. typhimurium TA98, but was activated in the presence of F5P/MPPT. The F5P/MPPT activated other polyaromatic hydrocarbons (PAH) in the S. typhimurium TA98 assay including dibenz[a,c]anthracene, dibenz[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene. The results indicated that the F5P/MPPT was the most efficient model for detecting PAH-induced mutagenicity in the Ames assay.

Lack of mutagenicity of diethylstilbestrol metabolite and analog, (±)-indenestrols A and B, in bacterial assays

Indenestrol A (IA), one of metabolites of the indanyl group of diethylstilbestrol, has a stronger binding affinity for the estrogen receptor and also a weaker uterotropic activity than endogenous estradiol. We tested the microbial mutagenicity of structural isomers of indenestrol A and indenestrol B (IB) in Salmonella typhimurium TA100 and TA98 and in Escherichia coli WP2 uvrA to investigate whether the interaction of diethylstilbestrol or IA with genomic DNA has any part in their carcinogenicity and other biological activities. In the absence of S9 mix, (+/-)-IA was cytotoxic at higher doses (1 and 10 mumol/plate), and both (+/-)-IA and (+/-)-IB were non-mutagenic at lower doses (0.1-100 nmol/plate). In the presence of S9 mix, (+/-)-IA was cytotoxic at higher doses (0.5 and 1 mumol/plate), and at the other doses, (+/-)-IA and (+/-)-IB did not show any distinct increase in revertants. Although (+/-)-IA and (+/-)-IB showed a slight increase in the revertants in strain TA100 by the preincubation method without S9 mix, these results were considered to be negative, because no reproducible dose-revertants relationship necessary for a chemical to be determined as mutagenic was obtained. The S9 fraction interacted with (+/-)-IA or (+/-)-IB enzymatically or non-enzymatically, and weakened its cytotoxicity, so that the toxic dose was higher in the presence of S9 mix than in its absence. Both the plate incorporation and preincubation methods were used with a wide range of concentrations of (+/-)-IA and (+/-)-IB in the present experiment. No clear positive mutagenic data were obtained. These results are the first reports on the mutation assays of (+/-)-IA and (+/-)-IB, and suggest that they were non-mutagenic towards the bacterial strains tested. The study revealed that the cytotoxic activity of (+/-)-IA and (+/-)-IB did not correlate with DNA interaction, but was the result of a direct effect on microtubule polymerization, although indenestrols are known to have strong binding affinities for estrogen receptors.

Selective proliferation of human γδ T cells in vitro

The effect of monoethylphosphate (MEP, commercial available or synthesized) together with IL-2 on the selective proliferation of human γδ T cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated. The γδ T cells were stimulated by MEP to proliferate in a dose-dependent manner. The effect of synthesized MEP was 10 times greater than that of commercial MEP. When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP, the total cell number increased about 1000-3000 fold; and the ratio of γδ T cells reached to 70-80%. The selective expansion of γδ T cells depended on the synergic action of MEP and IL-2. The bulk cultured γδ T cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5, K562 and Daudi) and autologous tumor cells. The culture system described here not only offers a simple method for obtaining a large number of γδ T cells which may become a new effector in the adoptive immunotherapy, but also provides a useful model for the further studies of the structure and function of γδ T cells in vitro.

Selective proliferation of human γ δ T cells in vitro

The effect of monoethylphosphate (MEP, commercial available or synthesized) together with IL-2 on the selective proliferation of human γδ T cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated. The γδ T cells were stimulated by MEP to proliferate in a dose-dependent manner. The effect of synthesized MEP was 10 times greater than that of commercial MEP. When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP, the total cell number increased about 1000-3000 fold; and the ratio of γδ T cells reached to 70-80%. The selective expansion of γδ T cells depended on the synergic action of MEP and IL-2. The bulk cultured γδ T cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5, K562 and Daudi) and autologous tumor cells. The culture system described here not only offers a simple method for obtaining a large number of γδ T cells which may become a new effector in the adoptive immunotherapy, but also provides a useful model for the further studies of the structure and function of γδ T cells in vitro.

Interprofessional education at the Keio University Faculty of Pharmacy

As part of a trial course on interprofessional education (IPE) at Keio University our Faculty of Pharmacy initiated a joint seminar with the university’s medical and nursing departments in 2008. We had two joint seminars: one in June and another in October. In the seminars, students and participants actively discussed several issues with regard to the national and private health care systems and medical malpractice. They also listened to feedback lectures held by experts from the medical and social system. At Kyoritsu University of Pharmacy, we had already held similar joint seminars three times since 2006. The participants were not only from our school but from various universities around Japan, given that our school was then only a small college for pharmaceutical students. Our considerable experience in the field of small group learning (SGL) helped make these seminars a success. By making full use of the SGL method we have successfully lead the discussion sessions at the IPE seminars attended by students from various medical fields. After the merging of our small pharmaceutical college with Keio University, we continued holding joint seminars for the three medical faculties at Keio University. It has not been without tribulations, however. The newly created interprofessional seminars faced several problems. The seeming lack of interest shown by the medical school decreased the level of participation of medical students, while too many facilitators are required for such seminars. To resolve these issues, we have made some changes to future activities in this area. To start with, we plan to change the course from an elective to a required subject for the students of the three medical faculties in Keio University.

Selective proliferation of human |[gamma]||[delta]| T cells in vitro

The effect of monoethylphosphate (MEP, commercial available or synthesized) together with IL-2 on the selective proliferation of human γδ T cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated. The γδ T cells were stimulated by MEP to proliferate in a dose-dependent manner. The effect of synthesized MEP was 10 times greater than that of commercial MEP. When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP, the total cell number increased about 1000-3000 fold; and the ratio of γδ T cells reached to 70-80%. The selective expansion of γδ T cells depended on the synergic action of MEP and IL-2. The bulk cultured γδ T cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5, K562 and Daudi) and autologous tumor cells. The culture system described here not only offers a simple method for obtaining a large number of γδ T cells which may become a new effector in the adoptive immunotherapy, but also provides a useful model for the further studies of the structure and function of γδ T cells in vitro.