phosphorus-radioisotopes and 2--deoxy-5--adenosine-monophosphate

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cattle; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Hydrogen-Ion Concentration; Kinetics; Phosphorus Radioisotopes; Solutions; Stereoisomerism

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Topics: Animals; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Exonucleases; Female; Isotope Labeling; Micrococcal Nuclease; Monocrotaline; Phosphorus Radioisotopes; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Reproducibility of Results; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Mass, Electrospray Ionization

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a, l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a, l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a, l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a, l]PDE-dAMP adduct was identified only by (32)P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a, l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.

Topics: Animals; Autoradiography; Benzopyrenes; Carcinogens; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Epoxy Compounds; Exonucleases; Micrococcal Nuclease; Microsomes, Liver; Phosphorus Radioisotopes; Rats; Spectrometry, Fluorescence; Stereoisomerism

Epoxybutanediol is one of the epoxide metabolites of butadiene (BD). A pair of diastereomeric N-1-adenine adducts were formed by reacting epoxybutanediol with deoxyadenosine 5'-monophosphate (5'-dAMP). These two N-1-adenine adducts rearranged in a base-catalysed reaction to an N6-trihydroxybutyl-adenine adduct, which was characterized by UV and mass spectroscopy. Using the 32P-postlabelling/HPLC assay the same adducts were detected in diepoxybutane (DEB)-treated DNA in vitro and in liver DNA samples from rats exposed to BD by inhalation. Adenine adducts of epoxybutanediol are probably suitable for monitoring BD exposure.

Topics: Animals; Butadienes; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; DNA Adducts; Epoxy Compounds; Phosphorus Radioisotopes; Rats

The concentrations of 2'-deoxyribonucleoside-3'-monophosphates remaining in calf-thymus DNA digests after nuclease P1 digestion or extraction into 1-butanol, the most commonly used adduct enrichment procedures prior to the application of the 32P-postlabelling assay, were measured using HPLC and 32P-postlabelling methods. When 10 micrograms of DNA digested to mononucleotides was used, the total amount of nucleotides remaining in the samples were approximately 4 and approximately 14 pmol after nuclease P1 treatment or 1-butanol extraction respectively. The influence of various concentrations of normal nucleotides on the labelling efficiency of a 2'-deoxyguanosine-3'-monophosphate adduct of benzo[a]pyrene diol-epoxide was also studied and found to depend upon the ratio of normal nucleotides/adducted nucleotides present in the sample. Also, the ATP/normal nucleotides ratio in the phosphorylation reaction may affect the quantitation of the adducts and thus deserves due consideration.

Topics: 1-Butanol; Animals; Benzo(a)pyrene; Butanols; Cattle; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; DNA; DNA Adducts; DNA Damage; Nucleotides; Phosphorus Radioisotopes; Single-Strand Specific DNA and RNA Endonucleases