2001 Fiscal Year Annual Research Report

(6-4)光産物からデュワー産物への光異性化を指標とした太陽光紫外線線量計の開発

Research Project

Project/Area Number	12558059
Research Institution	Kanazawa University
Principal Investigator	二階堂修金沢大学, 薬学部, 教授 (60019669)
Co-Investigator(Kenkyū-buntansha)	石垣靖人金沢大学, 薬学部, 助手 (20232275) 松永司金沢大学, 薬学部, 助教授 (60192340)
Keywords	損傷認識モノクローナル抗体 / デュワー型光産物 / ドットブロット解析 / 酸素標識免疫測定法(ELISA) / 太陽光紫外線 / UVB-UVA / UVC / リスク推定
Research Abstract	デュワー型光産物(DwP)は太陽光紫外線で誘発される二量体型損傷の一つであり、(6-4)光産物(64P)から太陽光紫外線に含まれる320nm前後の紫外線によって光異性化されて二次的に生じる。DwPは太陽光紫外線により細胞DNAに蓄積し、長く残存するためその変異原性が高いとされている。ヒトにおいてDwPに起因するリスクの推定には太陽光紫外線中に含まれる320nm付近の波長域の線量測定が必要である。しかし当該波長域(UVB-UVA境界域)の紫外線線量を測定する既存の機器は測定波長或の狭間にあるため無きに等しいのが現状である。従って本研究で開発する新しい太陽光紫外線線量計のリスク推定への寄与は大きいものと考えられる。 1.64Pの光異性化の波長依存性の検討予め100J/m^2のUVCを照射して64Pを誘発させておいたDNAに、国立岡崎共同研究機構・基礎生物学研究所に設置されている大型スペクトルグラフ装置(OLS)を用いて種々の波長の単色光紫外線を照射した。照射されたDNA標品を一本鎖化後、酵素標識免疫測定法(ELISA)にてDNA中の64P並びにDwPを測定した。64Pの測定には64M-2抗体を、DwP検出にはDEM-1抗体を一次抗体として用い検出した。その結果、DNA中に残存する64P量が低下するにつれてDwP量が増加する結果を得た。DWPの特異的誘発ピークは320nmにあり、64Pの示す特異的吸収波長に一致した。 2.免疫ドットブロット法(IDB)を利用した定量的損傷検出系(太陽光紫外線線量計)の樹立 Calf thymus DNAに100J/m^2のUVCを照射後、ナイロン膜(ロッシュ)にブロットし、フィルムバッジ様の太陽光受光器を製作した。次いでマイラーフィルターで300nm以下の波長をカットしたUVBランプ(東芝・FL-20SE)で種々の線量を照射した。線量測定には英弘精機のMI-2101を用いた。UVBを照射後、DwPを認識するDEM-1抗体でフィルターを処理し、アルカリフォスファターゼ標識ヤギ抗マウス抗体で2時間インキュベートした。次にCDP-star(ロッシュ)を滴下して発色させた。ナイロン膜上の化学発光シグナルは富士フイルムのLAS1000で撮影して画像解析し、Science Lab 98-Image Guage version 3によりバンド強度(band intensity)を定量化した。その結果、UVB照射線量に依存してバンド強度は直線的に増加することが明らかになった。 3.フィルムバッジ型測定器の屋外試験上記のフィルムバッジ型受光器は軽量で携帯に便である。しかし曝露される線量が高すぎると累積線量が正しく示さない可能性があるため、特異的波長吸収を示さないブルーポリエチレンフィルターをフィルムにかぶせることにより線量率を低下させることに成功した。その結果、金沢における日照量の最高日(初夏から夏にかけて)の紫外線線量に匹敵する線量をも正確に測ることが可能となった

Research Products
(22 results)

All Other

All Publications (22 results)

[Publications] Otoshi, E.: "Respective role of cyclobutane pyrimidine dimers, (6-4)photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair deficient xeroderma pigmentosum A cells"Cancer Res.. 60(6). 1729-1735 (2000)
[Publications] Ide, F.: "In vivo detection of ultraviolet photoproducts and their repair in Purkinje cells"Lab. Invest.. 80(4). 465-470 (2000)
[Publications] Horiki, S: "Protective effects of sunscreening agents on photocarcinogenesis, photoageing, and DNA damage in XPA gene knockout mice"Arch. Delmatol. Res.. 292(10). 511-518 (2000)
[Publications] Torizawa, T.: "DNA binding mode of the Fab fragment of a monoclonal antibody apecific for cyclobutane pyrimidine dimer"Nucleic Acids Res.. 28(4). 944-951 (2000)
[Publications] Perdis, D.: "Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammaliar cells. Possible role of Dewar Photoproducts in solar mutagenesis"J. Biol. Chem.. 275(35). 26732-26742 (2000)
[Publications] Yokoyama, H.: "Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultrviolet radiation"J. Mol. Biol.. 299(3). 711-723 (2000)
[Publications] Cario-Andre, M.: "Studies on epidermis reconstructed with and without melanocytes : Melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair-skinned caucasians"J. Invest. Dermatol.. 115(2). 193-199 (2000)
[Publications] Ray, AJ.: "The spectrum of mitochondrial DNA deletions in a ubiquitous marker of ultraviolet radiation exposure in human skin"J. Invest. Dermatol.. 115(4). 674-679 (2000)
[Publications] Takeuchi, S.: "Formation of DNA lesions in cucumber cotyledons exposed UV radiation"Environ. Sci.. 13. 351-355 (2000)
[Publications] Budiyanto, A.: "Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice"Carcinogenesis. 21(11). 2085-2090 (2000)
[Publications] Satou, K.: "Efficient chemical synthesis of a pyrimidine(6-4)pyrimidone photo-product analog and its properties"Tetrahedorn Letters. 41. 2175-2179 (2000)
[Publications] Sakamoto, A.: "Immuno expression of ultraviolet photoducts and p53 mutation analysis in atypical fibroxanthoma and superficial malignant fibrous histtiocytoma"Modern Patol.. 14. 581-588 (2001)
[Publications] Kiyosawa, K.: "Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Eschrichia coli recA mutant"Mutation Res.. 487. 149-156 (2001)
[Publications] Mone, M.J.: "Local UV-in duced DNA damage in cell nuclei results in local transcription inhibition"EMBO Rep. 2. 1013-1017 (2001)
[Publications] Katsumi, S.: "In situ visualization of ultraviolet-light-induced DNA damage repair in locally irradiated human fibroblasts"J. Ivnest. Dermatol.. 117. 1156-1161 (2001)
[Publications] Hayashi, S.: "The relationship between UVB svreening and cytoprotection by microcorpuscular ZnO or ascorbate against DNA photodamage and membrane injuries in keratinocyte by oxidative stress"J. Photochem. Photobiol., B.. 64. 27-35 (2001)
[Publications] Wakasugi, M.: "DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and RPA"J. Biol. Chem. 276. 15434-15440 (2001)
[Publications] Tanaka, M.: "Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichiacoli"Mutagenesis. 16(1). 1-6 (2001)
[Publications] Wakasugi, M.: "DDB accumulates at DNA damage sites immediately after UV-irradiation and directly stimulates nucleotide excision repair"J. Biol. Chem.. 227. 1637-1640 (2001)
[Publications] Ishigaki, Y.: "Evidence for a pioneer round of mRNA translation : mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20"Cell. 106. 607-617 (2001)
[Publications] 二階堂修: "太陽光紫外線とDNA"太陽紫外線防護研究委員会学術報告. 11. 47-59 (2001)
[Publications] Katayama, S.: "Fission yeast F-bok protein Pof3 is required for genomic instability and telomere function"Mol. Biol. Cell. 13. 211-224 (2002)

2001 Fiscal Year Annual Research Report

(6-4)光産物からデュワー産物への光異性化を指標とした太陽光紫外線線量計の開発

Principal Investigator

二階堂 修 金沢大学, 薬学部, 教授 (60019669)

Research Products

[Publications] Otoshi, E.: "Respective role of cyclobutane pyrimidine dimers, (6-4)photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair deficient xeroderma pigmentosum A cells"Cancer Res.. 60(6). 1729-1735 (2000)

[Publications] Ide, F.: "In vivo detection of ultraviolet photoproducts and their repair in Purkinje cells"Lab. Invest.. 80(4). 465-470 (2000)

[Publications] Horiki, S: "Protective effects of sunscreening agents on photocarcinogenesis, photoageing, and DNA damage in XPA gene knockout mice"Arch. Delmatol. Res.. 292(10). 511-518 (2000)

[Publications] Torizawa, T.: "DNA binding mode of the Fab fragment of a monoclonal antibody apecific for cyclobutane pyrimidine dimer"Nucleic Acids Res.. 28(4). 944-951 (2000)

[Publications] Perdis, D.: "Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammaliar cells. Possible role of Dewar Photoproducts in solar mutagenesis"J. Biol. Chem.. 275(35). 26732-26742 (2000)

[Publications] Yokoyama, H.: "Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultrviolet radiation"J. Mol. Biol.. 299(3). 711-723 (2000)

[Publications] Cario-Andre, M.: "Studies on epidermis reconstructed with and without melanocytes : Melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair-skinned caucasians"J. Invest. Dermatol.. 115(2). 193-199 (2000)

[Publications] Ray, AJ.: "The spectrum of mitochondrial DNA deletions in a ubiquitous marker of ultraviolet radiation exposure in human skin"J. Invest. Dermatol.. 115(4). 674-679 (2000)

[Publications] Takeuchi, S.: "Formation of DNA lesions in cucumber cotyledons exposed UV radiation"Environ. Sci.. 13. 351-355 (2000)

[Publications] Budiyanto, A.: "Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice"Carcinogenesis. 21(11). 2085-2090 (2000)

[Publications] Satou, K.: "Efficient chemical synthesis of a pyrimidine(6-4)pyrimidone photo-product analog and its properties"Tetrahedorn Letters. 41. 2175-2179 (2000)

[Publications] Sakamoto, A.: "Immuno expression of ultraviolet photoducts and p53 mutation analysis in atypical fibroxanthoma and superficial malignant fibrous histtiocytoma"Modern Patol.. 14. 581-588 (2001)

[Publications] Kiyosawa, K.: "Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Eschrichia coli recA mutant"Mutation Res.. 487. 149-156 (2001)

[Publications] Mone, M.J.: "Local UV-in duced DNA damage in cell nuclei results in local transcription inhibition"EMBO Rep. 2. 1013-1017 (2001)

[Publications] Katsumi, S.: "In situ visualization of ultraviolet-light-induced DNA damage repair in locally irradiated human fibroblasts"J. Ivnest. Dermatol.. 117. 1156-1161 (2001)

[Publications] Hayashi, S.: "The relationship between UVB svreening and cytoprotection by microcorpuscular ZnO or ascorbate against DNA photodamage and membrane injuries in keratinocyte by oxidative stress"J. Photochem. Photobiol., B.. 64. 27-35 (2001)

[Publications] Wakasugi, M.: "DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and RPA"J. Biol. Chem. 276. 15434-15440 (2001)

[Publications] Tanaka, M.: "Effects of photoreactivation of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts on ultraviolet mutagenesis in SOS-induced repair-deficient Escherichiacoli"Mutagenesis. 16(1). 1-6 (2001)

[Publications] Wakasugi, M.: "DDB accumulates at DNA damage sites immediately after UV-irradiation and directly stimulates nucleotide excision repair"J. Biol. Chem.. 227. 1637-1640 (2001)

[Publications] Ishigaki, Y.: "Evidence for a pioneer round of mRNA translation : mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20"Cell. 106. 607-617 (2001)

[Publications] 二階堂 修: "太陽光紫外線とDNA"太陽紫外線防護研究委員会学術報告. 11. 47-59 (2001)

[Publications] Katayama, S.: "Fission yeast F-bok protein Pof3 is required for genomic instability and telomere function"Mol. Biol. Cell. 13. 211-224 (2002)

二階堂修金沢大学, 薬学部, 教授 (60019669)

[Publications] 二階堂修: "太陽光紫外線とDNA"太陽紫外線防護研究委員会学術報告. 11. 47-59 (2001)