Grantee Research Project Results
Final Report: Solid-Matrix Luminescence Analysis of B[a]P-DNA Adducts
EPA Grant Number: R824100Title: Solid-Matrix Luminescence Analysis of B[a]P-DNA Adducts
Investigators: Hurtubise, Robert J.
Institution: University of Wyoming
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 1995 through September 30, 1998
Project Amount: $431,400
RFA: Exploratory Research - Chemistry and Physics of Water (1995) RFA Text | Recipients Lists
Research Category: Water , Land and Waste Management , Safer Chemicals
Objective:
The objectives of this research project were to develop the principles and methods for the solid-matrix luminescence characterization and determination of benzo[a]pyrene-DNA (B[a]P-DNA) adducts, for the capillary electrophoresis separation of benzo[a]pyrene metabolites, and for the laser-induced detection of benzo[a]pyrene metabolites and benzo[a]pyrene-DNA adducts in capillary electrophoresis.Summary/Accomplishments (Outputs/Outcomes):
The findings of this research are summarized below.
- Manuscripts. Several manuscripts overlapped with this project and a project that was funded previously by the U.S. Environmental Protection Agency (EPA) under grant number RA 817678. The manuscripts are cited at the end of this report (1-6).
- Solid-Matrix Luminescence Properties of Benzo[a]pyrene-DNA Adducts and Their Metabolites. Considerable data were acquired on the solid-matrix luminescence (SML) properties of the B[a]P-DNA adducts and their metabolites. In addition, several solid matrices were discovered that would permit SML to be obtained from B[a]P-DNA adducts and the metabolites. In particular, 30 percent TlNO3/sodium acetate (NaOAc) and Whatman 1PS paper were found to be excellent in obtaining solid-matrix phosphorescence (SMP) from B[a]P-DNA adducts and the metabolites. SMP lifetimes were acquired for (?)-anti-B[a]P-DNA, (+)-anti-B[a]P-DNA, and (-)-anti-B[a]P-DNA adsorbed on 30 percent TlNO3/NaOAc as a function of temperature. It was possible to identify the different forms of the adducts by their SMP lifetimes at low temperature. These are important results because the lifetimes can be employed not only to distinguish the adducts, but also tetrols, which are small molecular weight products of the adducts. In addition, it was demonstrated that the B[a]P-DNA adducts adsorbed on solid matrices were not highly sensitive to ultraviolet radiation. The adducts in solution are very sensitive to ultraviolet radiation (7,8).
- Several Methods for the Solid-Matrix Fluorescence and Solid-Matrix Phosphorescence Characterization of Benzo[a]pyrene-DNA Adducts with Whatman No. 1 and Whatman 1PS Papers. Both SMF and SMP were obtained from nanogram amounts of B[a]P-DNA adduct samples adsorbed on Whatman No. 1 and Whatman 1PS papers (9). Several SML methods were developed for the characterization of the adducts that were modified at relatively high levels (0.50, 0.60, and 0.98 percent) with these two types of paper and employing TlNO3 as a heavy-atom salt. Some of the methods developed were based on SMP selective excitation and moisture quenching, which allowed the detection of the quasi-intercalated adducts. Also, a unique method was developed for the SMF detection of B[a]P-DNA adducts that were externally bound to DNA by hydrophobic interaction of the adducts with 1PS paper (9).
- Determination of Benzo[a]pyrene-DNA Adducts by Solid-Matrix
Phosphorescence. One of the main goals of the research was to develop SMP
methods for the quantitation of B[a]P-DNA adducts. New SMP phosphorescence
methods were developed for the determination of the adducts at high levels of
modification and very low methods of modification. Initially, DNA was modified
at 2.6 percent, and both the SMF and SMP properties of the adducts were compared
on Whatman 1PS paper, Whatman 1PS paper with TlNO3, and 30 percent TlNO3/NaOAc.
The quantitative aspects of determining B[a]P-DNA adducts were explored by
comparing the SMF and SMP on these solid matrices. Calibration curves and limits
of detection were acquired with SMF for the B[a]P-DNA adducts adsorbed on the
three solid matrices. The SMP of the adducts were more intense on 30 percent
TlNO3/NaOAc than both the SMF and SMP of the adducts adsorbed on Whatman 1PS
paper. A typical linear range for the B[a]P-DNA adducts on 30 percent
TlNO3/NaOAc was 0 to 1.0 ng of adduct sample per mg of 30 percent TlNO3/NaOAc
with a limit of detection of 0.06 ng of B[a]P-DNA adduct per mg of 30 percent
TlNO3/NaOAc. In terms of molar concentration, the limit of detection with 30
percent TlNO3/NaOAc was 6 x 10-12 M. The methods developed would be beneficial
for researchers investigating B[a]P-DNA adducts from laboratory preparations
that are modified at relatively high levels (10).
One of the most important accomplishments in this research was the development of SMP methods for the SMP quantitation of B[a]P-DNA adducts at very low levels. It was demonstrated for the first time that linear relationships could be obtained between the SMP and percent modification of B[a]P-DNA adducts (11). Several samples of DNA were modified with the diolepoxide of benzo[a]pyrene (BPDE) at levels of 5.0 x 10-3, 1.0 x 10-3, 5.0 x 10-4, 1.0 x 10-4, 5.0 x 10-5, and 1.0 x 10-5. Data were obtained with both Whatman 1PS and 30 percent TlNO3/NaOAc solid matrices. The limit of detection of the B[a]P-DNA adducts was 2 adducts in 107 bases using both Whatman 1PS paper and 30 percent TlNO3/NaOAc. The previous limits of detection are at the level found in some human samples. It should be mentioned that there are very few methods for the direct determination of B[a]P-DNA adducts. The SMP method developed in this research is simple, does not use radioactive materials, and mild room-temperature conditions are used (11).
- Fluorescence of Tetrols, Tetrols Complexed with DNA, and Benzo[a]pyrene-DNA Adducts in Methanol:Water Solutions. In work partially supported by the EPA and Department of Energy (DOE), a rather detailed investigation was undertaken of the solution fluorescence properties of the four hydrolysis products of the B[a]P-DNA adducts (tetrols), tetrols complexed with DNA, and B[a]P-DNA adducts in methanol (MEOH):water solutions (12). The main reasons for performing this investigation were twofold: (1) MEOH:water solutions are used to deposit tetrols and B[a]P-DNA adducts on solid matrices, and thus, it is important to have detailed knowledge of the solution interactions prior to adsorbing the samples on solid matrices; and (2) the fluorescence properties of MeOH:water solutions of B[a]P-DNA adducts have not been studied in any detail, and therefore, it was suspected that some unique solute-solvent interactions would be revealed. The relative polarity of the environment for the tetrols and B[a]P-DNA adducts were determined by using a modified definition of the R-value that is commonly employed for pyrene. The R-value for the tetrols and the B[a]P-DNA adducts were calculated by obtaining the ratios of the intensities of the two major fluorescence emission bands at 380 nm and 400 nm. Methods were developed that showed unique interactions of tetrols with DNA and selective interactions of the B[a]P-DNA adducts with the MeOH-water solvents. In particular, the quasi-intercalated B[a]P-DNA adducts gave significant changes in the R-values with an increase in methanol in the solvent, and the excitation spectra showed large spectral shifts and changes in shape with an increase in methanol composition. The approaches developed provide very specific structural and polarity information on tetrols and B[a]P-DNA adducts.
- Capillary Electrophoresis of Polycyclic Aromatic Hydrocarbon
Metabolites. Several polycyclic aromatic hydrocarbon (PAH) metabolites were
investigated in this research. Some of them result from the metabolism of
B[a]P-DNA adducts and some of the metabolites occur from the metabolism of
B[a]P. Very little work has been done in the capillary electrophoresis (CE)
separation and characterization of the metabolites. In this research, CE methods
were developed to determine the pKa values of six important metabolites (13). In
our work, it was found that CE was very suitable for the determination of the
pKa values of weakly acidic, large ring PAH metabolites. Information on pKa
values is not only important in designing CE methods, but it is important in the
study of environmental toxins.
A major study was undertaken to develop highly efficient methods for the CE separation of PAH metabolites (14). A rapid CE method was developed using 3-(cyclohexylamino-1-propane sulfonic acid (CAPS) buffer for the compound-class separation of nine PAH metabolites, including tetrols, benzo[a]pyrene diols, and hydroxyl aromatics. Also, a detailed investigation was performed on the effects of organic solvents on the electroosmotic flow and electrophoretic mobilities of the analytes. The separation of the nine PAH metabolites was achieved within 24 minutes with 100 mM CAPS buffer in 40 percent (v/v) MeOH at a pH of 10.4. In addition, it was possible to partially resolve the parent PAH compounds from metabolites with the previous buffer system. The simple CE methods developed would be useful for the rapid separation and characterization of several important biomarkers. The CE methods developed are simple and easy to use, and the new methodology should find a wide range of applications for similar PAH metabolites.
- Separation of Polycyclic Aromatic Hydrocarbon Metabolites by -Cyclodextrin-Modified Micellar Electrokinetic Chromatography. After a detailed investigation, a -cyclodextrin ( -CD) modified micellar electrokinetic capillary chromatographic (CD-MEKC) method was developed to separate PAH metabolites (15). Baseline resolution of six acidic PAH metabolites was achieved within 14 minutes. The effects of several separation parameters were investigated in detail. These were the -CD concentration, sodium dodecyl sulfate concentration, pH of the buffer, organic solvent modifiers, and urea concentration. The apparent capacity factor increased linearly with sodium dodecyl sulfate concentration, which was consistent with theory. Possible mechanisms that account for the differences in the apparent distribution coefficients were proposed. The CD-MEKC method was compared with the CE method discussed in the previous section. For the separation of the six acidic metabolites, the CD-MEKC system was the better method in terms of separation efficiency and sensitivity.
- Capillary Electrophoresis of Carcinogenic Heterocyclic Aromatic Amines. In work that was partially supported by DOE and EPA, CE methods were developed for the separation of toxic heterocyclic aromatic amines (HAA) that are found in foods (16). Also, CE methods were developed for the determination of the ionization constants of the HAA (17). These are an important class of toxic compounds that can form DNA adducts and are highly suspect in causing cancer.
Journal Articles on this Report : 15 Displayed | Download in RIS Format
Other project views: | All 25 publications | 15 publications in selected types | All 15 journal articles |
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Type | Citation | ||
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Chu Y, Hurtubise RJ. Comparison of the solid-matrix luminescence properties of Benzo(a)Pyrene-DNA adducts on alpha-cyclodextrin/NaCl and trehalose/NaCl matrices. Photochemistry and Photobiology 1995;62(2):251-257. |
R824100 (Final) |
not available |
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Chu Y, Hurtubise RJ. Effects of a heavy-atom salt on the solid-matrix luminescence properties of the (+/-) trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene-DNA adducts and tetrol I-1 adsorbed on several solid matrices. Applied Spectroscopy 1996;50(4):476-482. |
R824100 (Final) |
not available |
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Li M, Hurtubise RJ. Characterization and quantitation of benzo[a]pyrene-DNA adducts by solid-matrix luminescence. Analytical Letters 1998;31(3):491-510. |
R824100 (1998) R824100 (Final) |
not available |
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Li M, Hurtubise RJ, Weston A. Determination of benzo[a]pyrene-DNA adducts by solid- matrix phosphorescence. Analytical Chemistry 1999;71(20):4679-4683 |
R824100 (Final) |
not available |
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Mendonsa SD, Hurtubise RJ. Determination of ionization constants of heterocyclic aromatic amines using capillary zone electrophoresis. Journal of Chromatography A 1999;841(2):239-247. |
R824100 (Final) |
not available |
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Mendonsa SD, Hurtubise RJ. Capillary electrophoresis (CE) methods for the separation of carcinogenic heterocyclic aromatic amines. Journal of Liquid Chromatography & Related Technologies 1999;22(7):1027-1040. |
R824100 (Final) |
not available |
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Ramasamy SM, Hurtubise RJ, Weston A. Detection of 1-hydroxypyrene as a urine biomarker of human PAH exposure determined by fluorescence and solid-matrix luminescence spectroscopy. Applied Spectroscopy 1997;51(3):1377-1383. |
R824100 (Final) |
not available |
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Rozbeh M, Hurtubise RJ. Reversed-phase chromatographic separation of benzo[a]pyrene metabolites with beta-cyclodextrin as a mobile phase-additive. Journal of Liquid Chromatography 1995;18(1):17-37. |
R824100 (Final) |
not available |
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Rozbeh M, Hurtubise RJ. The liquid-chromatographic separation of metabolites of benzo[a]pyrene with gamma-cyclodextrin as a mobile-phase additive. Journal of Liquid Chromatography 1995;18(10):1909-1931. |
R824100 (Final) |
not available |
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Steinbach PB, Hurtubise RJ. Fluorescence of tetrols, tetrols complexed with DNA, and benzo[a]pyrene-DNA adducts in methanol/water solutions. Applied Spectroscopy 2000;54(2):287-293. |
R824100 (Final) |
not available |
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Tjioe SW, Ramasamy SM, Hurtubise RJ. Effects of ultraviolet light on r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adducts adsorbed on filter paper and the detection of tetrols by synchronous solid-matrix phosphorescence. Analytical Letters 1997;30(5):985-999. |
R824100 (Final) |
not available |
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Tjioe SW, Hurtubise RJ. Solid-matrix fluorescence and phosphorescence detection and characterization of benzo[a]pyrene-DNA adducts with Whatman 1 and Whatman 1PS filter paper. Applied Spectroscopy 1998;52(3):414-419. |
R824100 (1998) R824100 (Final) |
not available |
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Xu X, Hurtubise RJ. Influence of organic solvents in the capillary zone electrophoresis of polycyclic aromatic hydrocarbon metabolites. Journal of Chromatography A 1998;(1-2):289-299. |
R824100 (Final) |
not available |
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Xu X, Hurtubise RJ. Determination of the pK(a) values of polycyclic aromatic hydrocarbon metabolites by capillary zone electrophoresis. Journal of Liquid Chromatography & Related Technologies 1999;22(5):669-679. |
R824100 (1998) R824100 (Final) |
not available |
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Xu X, Hurtubise RJ. Separation of polycyclic aromatic hydrocarbon metabolites by gamma-cyclodextrin-modified micellar electrokinetic chromatography. Journal of Liquid Chromatography & Related Technologies 2000;23(11):1657-1670. |
R824100 (Final) |
not available |
Supplemental Keywords:
property values, environmental damage, stigma., Health, Scientific Discipline, Water, Physics, Environmental Chemistry, Chemistry, Risk Assessments, Engineering, Engineering, Chemistry, & Physics, sediment toxicity, benzopyrene, human exposure, electrophoretic studies, soil contaminants, DNA adducts, solid matrix luminescence, physicochemical, heavy atom effects, capillary zoneProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.