Identification and quantitative detection of isomeric benzo[a]pyrene diolepoxide-DNA adducts by low-temperature conventional fluorescence methods

Rushen Zhao, Tong Ming Liu, Seog K. Kim, Michael C. MacLeod, Nicholas Geacintov

Research output: Contribution to journalArticle

Abstract

The pyrene-like fluorescence of adducts derived from the covalent binding of (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] to DNA increases in intensity by factors of 20 or more as the temperature is lowered from ambient to ∼100 K. This effect is primarily associated with the strong quenching of the pyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electron-transfer quenching mechanism at 100 K. In contrast, the fluorescence of BPDE-deoxyadenosyl adducts is not quenched at ambient temperatures, and the fluorescence yields of (±)-anti-BPDE-poly(dA-dT)·(dA-dT) adducts increases by only a factor of 2 in this same temperature range. Utilizing an internal fluorescein fluorescence standard to correct for differences in light scattering and variations in instrumental factors, a fluorescence method is described for quantitatively determining the levels of benzo[a]pyrene diolepoxide derivatives covalently bound to cellular DNA at 100 K. The method is illustrated with (±)-reverse-BPDE [(±)-trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene]. Adduct levels as low as 10 pmol in a 400 μl sample volume can be detected and identified from their excitation and fluorescence emission spectra using a conventional and commercially available fluorometer. In the case of modified DNA extracted from BPDE-treated Chinese hamster ovary cells or from mouse skin (∼1 BPDE residue/20 000 bases), such an analysis requires only 100 μg of DNA. Conformationally different adducts derived from the binding of the isomeric (±)-anti-BPDE, (±)-reverse-BPDE or (±)-syn-BPDE to cellular DNA can be distinguished by their low-temperature fluorescence excitation spectra. Specifically, the quasi-intercalated site I BPDE adducts (believed to be associated with cis-addition stereochemistry) can be distinguished from site II adducts situated at external BPDE binding sites (trans-addition stereochemistry). These results suggest that the fates of these conformationally different BPDE-DNA adducts, e.g. due to enzymatic repair, can be monitored as a function of time in DNA extracted from intact, functioning cells.

Original languageEnglish (US)
Pages (from-to)1817-1824
Number of pages8
JournalCarcinogenesis
Volume13
Issue number10
StatePublished - Oct 1992

Fingerprint

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Pyrene
Fluorescence
Temperature
DNA
Stereochemistry
Epoxy
Quenching
Reverse
Excitation
Fluorometers
Electron Transfer
Ovary
DNA Adducts
benzo(a)pyrene-DNA adduct
Cell
Light Scattering
Poly dA-dT
Binding sites
Light scattering

ASJC Scopus subject areas

  • Cancer Research
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Physiology
  • Behavioral Neuroscience

Cite this

Identification and quantitative detection of isomeric benzo[a]pyrene diolepoxide-DNA adducts by low-temperature conventional fluorescence methods. / Zhao, Rushen; Liu, Tong Ming; Kim, Seog K.; MacLeod, Michael C.; Geacintov, Nicholas.

In: Carcinogenesis, Vol. 13, No. 10, 10.1992, p. 1817-1824.

Research output: Contribution to journalArticle

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title = "Identification and quantitative detection of isomeric benzo[a]pyrene diolepoxide-DNA adducts by low-temperature conventional fluorescence methods",
abstract = "The pyrene-like fluorescence of adducts derived from the covalent binding of (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] to DNA increases in intensity by factors of 20 or more as the temperature is lowered from ambient to ∼100 K. This effect is primarily associated with the strong quenching of the pyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electron-transfer quenching mechanism at 100 K. In contrast, the fluorescence of BPDE-deoxyadenosyl adducts is not quenched at ambient temperatures, and the fluorescence yields of (±)-anti-BPDE-poly(dA-dT)·(dA-dT) adducts increases by only a factor of 2 in this same temperature range. Utilizing an internal fluorescein fluorescence standard to correct for differences in light scattering and variations in instrumental factors, a fluorescence method is described for quantitatively determining the levels of benzo[a]pyrene diolepoxide derivatives covalently bound to cellular DNA at 100 K. The method is illustrated with (±)-reverse-BPDE [(±)-trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene]. Adduct levels as low as 10 pmol in a 400 μl sample volume can be detected and identified from their excitation and fluorescence emission spectra using a conventional and commercially available fluorometer. In the case of modified DNA extracted from BPDE-treated Chinese hamster ovary cells or from mouse skin (∼1 BPDE residue/20 000 bases), such an analysis requires only 100 μg of DNA. Conformationally different adducts derived from the binding of the isomeric (±)-anti-BPDE, (±)-reverse-BPDE or (±)-syn-BPDE to cellular DNA can be distinguished by their low-temperature fluorescence excitation spectra. Specifically, the quasi-intercalated site I BPDE adducts (believed to be associated with cis-addition stereochemistry) can be distinguished from site II adducts situated at external BPDE binding sites (trans-addition stereochemistry). These results suggest that the fates of these conformationally different BPDE-DNA adducts, e.g. due to enzymatic repair, can be monitored as a function of time in DNA extracted from intact, functioning cells.",
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T1 - Identification and quantitative detection of isomeric benzo[a]pyrene diolepoxide-DNA adducts by low-temperature conventional fluorescence methods

AU - Zhao, Rushen

AU - Liu, Tong Ming

AU - Kim, Seog K.

AU - MacLeod, Michael C.

AU - Geacintov, Nicholas

PY - 1992/10

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N2 - The pyrene-like fluorescence of adducts derived from the covalent binding of (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] to DNA increases in intensity by factors of 20 or more as the temperature is lowered from ambient to ∼100 K. This effect is primarily associated with the strong quenching of the pyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electron-transfer quenching mechanism at 100 K. In contrast, the fluorescence of BPDE-deoxyadenosyl adducts is not quenched at ambient temperatures, and the fluorescence yields of (±)-anti-BPDE-poly(dA-dT)·(dA-dT) adducts increases by only a factor of 2 in this same temperature range. Utilizing an internal fluorescein fluorescence standard to correct for differences in light scattering and variations in instrumental factors, a fluorescence method is described for quantitatively determining the levels of benzo[a]pyrene diolepoxide derivatives covalently bound to cellular DNA at 100 K. The method is illustrated with (±)-reverse-BPDE [(±)-trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene]. Adduct levels as low as 10 pmol in a 400 μl sample volume can be detected and identified from their excitation and fluorescence emission spectra using a conventional and commercially available fluorometer. In the case of modified DNA extracted from BPDE-treated Chinese hamster ovary cells or from mouse skin (∼1 BPDE residue/20 000 bases), such an analysis requires only 100 μg of DNA. Conformationally different adducts derived from the binding of the isomeric (±)-anti-BPDE, (±)-reverse-BPDE or (±)-syn-BPDE to cellular DNA can be distinguished by their low-temperature fluorescence excitation spectra. Specifically, the quasi-intercalated site I BPDE adducts (believed to be associated with cis-addition stereochemistry) can be distinguished from site II adducts situated at external BPDE binding sites (trans-addition stereochemistry). These results suggest that the fates of these conformationally different BPDE-DNA adducts, e.g. due to enzymatic repair, can be monitored as a function of time in DNA extracted from intact, functioning cells.

AB - The pyrene-like fluorescence of adducts derived from the covalent binding of (±)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] to DNA increases in intensity by factors of 20 or more as the temperature is lowered from ambient to ∼100 K. This effect is primarily associated with the strong quenching of the pyrene-like fluorescence of BPDE-deoxyguanosyl adducts at room temperature, and the suppression of the electron-transfer quenching mechanism at 100 K. In contrast, the fluorescence of BPDE-deoxyadenosyl adducts is not quenched at ambient temperatures, and the fluorescence yields of (±)-anti-BPDE-poly(dA-dT)·(dA-dT) adducts increases by only a factor of 2 in this same temperature range. Utilizing an internal fluorescein fluorescence standard to correct for differences in light scattering and variations in instrumental factors, a fluorescence method is described for quantitatively determining the levels of benzo[a]pyrene diolepoxide derivatives covalently bound to cellular DNA at 100 K. The method is illustrated with (±)-reverse-BPDE [(±)-trans-9,10-dihydroxy-anti-7,8-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene]. Adduct levels as low as 10 pmol in a 400 μl sample volume can be detected and identified from their excitation and fluorescence emission spectra using a conventional and commercially available fluorometer. In the case of modified DNA extracted from BPDE-treated Chinese hamster ovary cells or from mouse skin (∼1 BPDE residue/20 000 bases), such an analysis requires only 100 μg of DNA. Conformationally different adducts derived from the binding of the isomeric (±)-anti-BPDE, (±)-reverse-BPDE or (±)-syn-BPDE to cellular DNA can be distinguished by their low-temperature fluorescence excitation spectra. Specifically, the quasi-intercalated site I BPDE adducts (believed to be associated with cis-addition stereochemistry) can be distinguished from site II adducts situated at external BPDE binding sites (trans-addition stereochemistry). These results suggest that the fates of these conformationally different BPDE-DNA adducts, e.g. due to enzymatic repair, can be monitored as a function of time in DNA extracted from intact, functioning cells.

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