Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically and by gamma radiation.

Coumarin-3-carboxylic acid (3-CCA) was used as a detector for hydroxyl radicals (.OH) in aqueous solution. The .OH was generated by gamma irradiation or chemically by the Cu2+-mediated oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by gamma irradiation, were nearly identical to those of an authentic 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). The pH-titration curves for the fluorescence at 450 nm (excitation at 395 nm) of 3-CCA, hydroxylated either chemically or by gamma radiation, were also identical to those of authentic 7-OHCCA (pK = 7.4). Time-resolved measurements of the fluorescence decays of radiation- or chemically hydroxylated 3-CCA, as well as those of 7-OHCCA, indicate a monoexponential fit. The fluorescence lifetime for the product of 3-CCA hydroxylation was identical to that of 7-OHCCA (approximately 4 ns). These data, together with analysis of end products by high-performance liquid chromatography, show that the major fluorescent product formed by radiation-induced or chemical hydroxylation of 3-CCA is 7-OHCCA. Fluorescence detection of 3-CCA hydroxylation allows real-time measurement of the kinetics of .OH generation. The kinetics of 3-CCA hydroxylation by gamma radiation is linear, although the kinetics of 3-CCA hydroxylation by the Cu2+-ASC reaction shows a sigmoid shape. The initial (slow) step of 3-CCA hydroxylation is sensitive to Cu2+, but the steeper (fast) step is sensitive to ASC. Analysis of the kinetics of 3-CCA hydroxylation shows a diffusion-controlled reaction with a rate constant 5.0 +/- 1.0 x 10(9) M(-1) s(-1). The scavenging of .OH by 3-CCA was approximately 14% for chemical generation with Cu2+-ASC and approximately 50% for gamma-radiation-produced .OH. The yield of 7-OHCCA under the same radiation conditions was approximately 4.4% and increased linearly with radiation dose. The 3-CCA method of detection of .OH is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems.

[1]  Stewart A. Brown,et al.  The natural coumarins : occurrence, chemistry, and biochemistry , 1982 .

[2]  A. Grollman,et al.  Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG , 1991, Nature.

[3]  A. Kassis,et al.  Quantification of radiation-induced hydroxyl radicals within nucleohistones using a molecular fluorescent probe. , 1994, Radiation research.

[4]  K D Held,et al.  Radiation-induced apoptosis in HL60 cells: oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose. , 1996, Radiation research.

[5]  A. Martell,et al.  Metal ion and metal chelate catalyzed oxidation of ascorbic acid by molecular oxygen. II. Cupric and ferric chelate catalyzed oxidation. , 1967, Journal of the American Chemical Society.

[6]  P. Fromherz.  Lipid coumarin dye as a probe of interfacial electrical potential in biomembranes. , 1989, Methods in enzymology.

[7]  M. Grisham.  Reactive metabolites of oxygen and nitrogen in biology and medicine , 1992 .

[8]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[9]  J Biaglow,et al.  Benzoyl peroxide acts as a promoter of radiation induced malignant transformation in vitro. , 1995, Biochemical and biophysical research communications.

[10]  R. T. Williams,et al.  Studies in detoxication. 72. The metabolism of coumarin and of o-coumaric acid. , 1958, The Biochemical journal.

[11]  C. Sonntag,et al.  The chemical basis of radiation biology , 1987 .

[12]  K. Held,et al.  Role of guanosine triphosphate in ferric ion-linked Fenton chemistry. , 1996, Radiation research.

[13]  A. Weissberger,et al.  Oxidation Processes. XVII.1 The Autoxidation of Ascorbic Acid in the Presence of Copper , 1944 .

[14]  R. Grinstead.  The Oxidation of Ascorbic Acid by Hydrogen Peroxide. Catalysis by Ethylenediaminetetraacetato-Iron(III) , 1960 .

[15]  B. Halliwell,et al.  Detection of hydroxyl radicals by aromatic hydroxylation. , 1994, Methods in enzymology.

[16]  S. Adelstein,et al.  Radiation damage to histone H2A by the primary aqueous radicals. , 1987, Radiation research.

[17]  R. H. Goodwin,et al.  The fluorescence of coumarin derivatives as a function of pH. II. , 1952, Archives of biochemistry and biophysics.

[18]  O. Aruoma.  [5] Deoxyribose assay for detecting hydroxyl radicals , 1994 .

[19]  X. Shen,et al.  Copper catalyzed oxidation of ascorbate: chemical and ESR studies. , 1990, Lens and eye toxicity research.

[20]  K. Held,et al.  The importance of peroxide and superoxide in the X-ray response. , 1992, International journal of radiation oncology, biology, physics.

[21]  D. Crosby,et al.  Fluorescence spectra of some simple coumarins. , 1962, Analytical biochemistry.

[22]  M. Flick,et al.  Radiation chemical mechanisms of single- and double-strand break formation in irradiated SV40 DNA. , 1991, Radiation research.

[23]  B. Halliwell,et al.  Interactions of a series of coumarins with reactive oxygen species. Scavenging of superoxide, hypochlorous acid and hydroxyl radicals. , 1992, Biochemical pharmacology.

[24]  M. Tien,et al.  COMPARATIVE ASPECTS OF SEVERAL MODEL LIPID PEROXIDATION SYSTEMS , 1982 .

[25]  K. Held,et al.  Quantitation of hydroxyl radicals produced by radiation and copper-linked oxidation of ascorbate by 2-deoxy-D-ribose method. , 1997, Free radical biology & medicine.

[26]  E. Robins,et al.  Fluorescence of substituted 7-hydroxycoumarins. , 1968, Analytical chemistry.

[27]  D. T. Sawyer,et al.  Does superoxide ion oxidize catechol, .alpha.-tocopherol, and ascorbic acid by direct electron transfer? , 1980 .

[28]  A. Kassis,et al.  A method for detection of hydroxyl radicals in the vicinity of biomolecules using radiation-induced fluorescence of coumarin. , 1993, International journal of radiation biology.

[29]  八木 国夫.  Lipid peroxides in biology and medicine , 1982 .

[30]  R. Weichselbaum,et al.  Coinduction of c-jun gene expression and internucleosomal DNA fragmentation by ionizing radiation. , 1993, Biochemistry.

[31]  R. D. Murray.  The natural coumarins , 1982 .

[32]  U. Madhvanath,et al.  The aqueous coumarin system as a low range chemical dosimeter , 1979 .

[33]  G K Svensson,et al.  Coumarin chemical dosimeter for radiation therapy. , 1994, Medical physics.

[34]  E. Gajewski,et al.  Structure and mechanism of hydroxyl radical-induced formation of a DNA-protein cross-link involving thymine and lysine in nucleohistone. , 1989, Cancer research.

[35]  P. Fromherz,et al.  Interfacial pH at electrically charged lipid monolayers investigated by the lipoid pH-indicator method. , 1974, Biochimica et biophysica acta.

[36]  B. Brodie,et al.  Ascorbic acid in aromatic hydroxylation. II. Products formed by reaction of substrates with ascorbic acid, ferrous ion, and oxygen. , 1954, The Journal of biological chemistry.

[37]  J. Gutteridge,et al.  Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants. , 1983, Biochimica et biophysica acta.

[38]  K. Schilling,et al.  Oxygen effect in the radiolysis of proteins. Part 2. Bovine serum albumin. , 1984, International journal of radiation biology and related studies in physics, chemistry, and medicine.