phosphorus-radioisotopes and styrene-oxide

A 32P-postlabeling method was established for the quantitative characterization of 2'-deoxyguanosyl O6-adducts of styrene 7,8-oxide in DNA. The two regioisomeric adducts, O6-(2-hydroxyl-1-phenylethyl)-2'-deoxyguanosine 3'-phosphate (alpha-isomer) and O6-(2-hydroxyl-2-phenylethyl)-2'-deoxyguanosine 3'-phosphate (beta-isomer), were synthesized and used for optimizing and quantifying the various analytical steps. The adducts were stable at pH 7 and 10, but not at pH 4. The adducts were sensitive to dephosphorylation during the standard nuclease P1 (NP1) treatment. Within 30 min, 73 and 94% of the alpha- and beta-isomers were digested. Adducts could not be extracted into butanol, and micropreparative chromatography on reversed-phase thin layers resulted in a loss of adducts at low levels. Therefore, further methods of enrichment had to be investigated. Micropreparative reversed-phase HPLC chromatography on a C18 column resulted in a many thousand-fold purification from the normal nucleotides. Further enrichment was achieved with a mild NP1 treatment. The phosphorylation efficiency with polynucleotide kinase was 5 and 15% for the alpha- and beta-isomers, respectively. Adduct analysis was performed with reversed-phase TLC followed by contact transfer of the origin to a polyethyleneimine-cellulose sheet and two-dimensional development. Addition of various amounts of adduct standard to the hydrolysate of 30 microg of DNA isolated from a control rat liver showed limits of detection of three and two adducts per 10(7) nucleotides for the alpha- and beta-isomers, respectively. The applicability of the newly developed method was demonstrated by the DNA analysis of styrene-exposed rats.

Topics: Adenosine Triphosphate; Animals; Carcinogens; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; DNA Adducts; Epoxy Compounds; Female; Guanine; Hydrogen-Ion Concentration; Isotope Labeling; Liver; Magnetic Resonance Spectroscopy; Phosphorus Radioisotopes; Rats; Single-Strand Specific DNA and RNA Endonucleases; Spectrophotometry, Ultraviolet

Styrene 7,8-oxide, which reacts preferentially at the N-7 position of guanine, yielded two pairs of diastereomers 7-(1'-hydroxy-2'-phenylethyl)-dGMP (the alpha-isomer) and 7-(2-hydroxy-2-phenylethyl)-dGMP (the beta-isomer) on reaction with deoxyguanosine-3'-monophosphate (3'-dGMP). The alpha- and beta-isomers were formed in the ratio 32:68. T4 polynucleotide kinase preferentially mediated labelling of diastereomers corresponding to the beta-isomer. The beta-diastereomers showed a labelling efficiency of 52%, whereas the alpha-isomers showed a labelling efficiency of 4%. Molecular modelling experiments showed intrinsic differences between the two isomers. The torsion angles of C8-N7-2'-Ar and C8-N7-2'-1' for the alpha-isomers were 149.9 degrees and -26.4 degrees, whereas the torsion angles of C-8-N7-1'-2' and N7-1'-2'-Ar for the beta-isomers were 105.3 degrees and 179.4 degrees. The consequent interatomic distance between one of the hydrogens on the alpha-carbon and the 3'-phosphate group on the sugar residue was 5.3 A in the alpha-isomer whereas the closest distance between the hydrogens attached to the alpha-carbon and 3'-phosphate group in the beta-isomer was 6.2 A. This arrangement probably leads to steric overcrowding at the 3'-phosphate group in alpha-isomers and these are less efficiently phosphorylated than beta-isomers. In in vitro styrene oxide-modified salmon testis DNA alpha- and beta-isomers of 7-alkylguanines were formed in the ratio 37:63. The recovery of two diastereomeric beta-isomers in a 32P-postlabelling assay was 14%, but one of the diastereomers was obtained in 3-fold greater yield than the second isomer.

Topics: Animals; Carcinogens; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; DNA; Epoxy Compounds; Male; Models, Chemical; Phosphorus Radioisotopes; Phosphorylation; Stereoisomerism

The modified 32P-postlabeling method was used for the detection of N-7-(2-hydroxy-phenylethyl) guanine adducts in DNA and human embryonal lung (HEL) cells treated in vitro with styrene oxide (SO). The total recovery of 7-alkylguanine adducts of styrene oxide in DNA analysed by 32P-postlabeling assay was 4.1 +/- 0.6%. The disappearance of 7-alkyldeoxyguanosine monophosphate adducts from SO-modified DNA at 37 degrees C showed a half-life of 19 h. The levels of 7-alkylguanine DNA adducts and single-strand breaks (SSBs) in DNA were determined in HEL cells treated with SO for 3 and 18 h. In the 3 h treatment there was a concentration-dependent increase of both 7-alkylguanine adducts and SSBs in DNA (r = 0.98, P = 0.012 and r = 0.99, P = 0.003, respectively). We found a significant correlation between 7-alkylguanine DNA adducts and SSBs in DNA (r = 0.98, P = 0.011). In HEL cells approximately 3-fold higher levels of both 7-alkylguanine DNA adducts and SSB in DNA were found after the SO (100 microM) treatment for 3 h than after the treatment for 18 h. A significant concentration-dependent increase was found only for SSB ( r = 0.95, P = 0.024) in the 18 h treatment with SO. There was no significant correlation between 7-alkylguanine adducts and SSBs (r = 0.72, P = 0.15). Our data suggest relatively fast removal of both 7-alkylguanine adducts of SO and DNA SSBs.

Topics: Autoradiography; Cell Line; Chromatography, High Pressure Liquid; DNA Adducts; DNA, Single-Stranded; Epoxy Compounds; Humans; Lung; Mutagens; Phosphorus Radioisotopes; Spectrophotometry, Ultraviolet

32P-Post-labeling was used to analyze for the presence of DNA adducts in 47 workers exposed to styrene in a boat manufacturing facility. Individual airborne exposures measured several times over the course of 1 year ranged from 1 to 235 mg/m3 with a mean value of 65.6 mg/m3. Two adducts were detected in the DNA of mononuclear cells of these workers. The following levels of adducts were detected: adduct 1, range 0.6-102 x 10(-8) (mean 15.8 x 10(-8); adduct 2, range 0.1-70.9 x 10(-8) (mean 14.2 x 10(-8). Significant linear relationships were found between styrene exposure and both DNA adducts (adduct 2, r = 0.330, P = 0.012; adduct 1, r = 0.244, P = 0.049). Co-chromatography experiments identified DNA adduct 1 in the exposed samples as N2-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3',5'-bisphosphate. DNA adduct 2 remains unidentified. No significant linear relationships were observed between the level of DNA adducts and sister chromatid exchanges, possibly because of the poor precision of the 32P-post-labeling assay (the estimated coefficients of variation for adducts 1 and 2 were 2.54 and 1.96, respectively). These results demonstrate that occupational exposure to styrene results in the formation of DNA adducts in human mononuclear cells.

Topics: DNA; Epoxy Compounds; Female; Humans; Leukocytes, Mononuclear; Male; Occupational Exposure; Phosphorus Radioisotopes; Sister Chromatid Exchange; Styrene; Styrenes

Lamination workers are exposed to large amounts of styrene. A postlabelling method was developed in order to detect DNA adducts in white blood cells from such workers. First, styrene oxide was reacted with DNA, and the adducts were characterized by the nuclease P1 version of the 32P-postlabelling technique. The adducts in human samples were transferred magnetically during chromatography. The 2'-deoxyguanosine 3'-monophosphate (dGMP) adduct standards, including N2 and O6 adducts, were shown to be resistant to the action of nuclease P1. The O6 adducts were detected in the femtomole range at about 10% labelling efficiency. In lamination workers, the level of O6 adducts, adjusted for adduct recovery, was 5/10(8) nucleotides, which was five times the level in controls.

Topics: Autoradiography; Chromatography, Thin Layer; Deoxyguanine Nucleotides; DNA; Epoxy Compounds; Humans; Occupational Exposure; Phosphorus Radioisotopes; Single-Strand Specific DNA and RNA Endonucleases

Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P1-enriched adducts were 32P-labelled and purified by high-salt (4.0 M ammonium formate, pH 6.1) C18 reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H2O (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions ([gamma-32P]ATP, < or = 3000 Ci/mmol; < or = 2 microM) resulted in poor (4-7%) adduct recovery. An ATP concentration of 40 microM, however, increased the labelling efficiency by a factor of 5-8 (35-55%) based on 3H-SO labelled DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved.

Topics: Animals; Carcinogens; Chromatography, Thin Layer; DNA; DNA Damage; Epoxy Compounds; Evaluation Studies as Topic; In Vitro Techniques; Male; Mutagens; Phosphorus Radioisotopes

Adducts were prepared by reacting styrene oxide with 2-deoxyguanosine 3'-monophosphate (dGMP). Four isomeric N-7-, two diastereomeric N2- and three isomeric O6-adduct were isolated and characterized. The adducts were used as substrates in the 32P-postlabeling reaction. No phosphorylation products were seen with the N-7-alkylation products. One diastereomeric N2-adduct was labeled with 20% efficiency and the second with a markedly lower efficiency. Two of the three O6-adducts were labeled with 5% and the third with 10% labeling efficiency. The results suggest that large N-7-dGMP adducts are very poor substrates of T4 polynucleotide kinase. The diastereomeric products are labeled at different efficiencies indicating stereoselectivity in the kinase reaction.

Topics: Deoxyguanine Nucleotides; Epoxy Compounds; Phosphorus Radioisotopes; Phosphorylation; Polynucleotide 5'-Hydroxyl-Kinase; Stereoisomerism

32P post-labeling of DNA reacted with styrene oxide resulted in the detection of six adducts. In order to determine which of these corresponded to modification at the O6 position of guanine, O6-substituted styrene oxide-deoxyguanosine-3'-monophosphate derivatives were synthesized. The two synthetic isomers were purified by HPLC and the structures were confirmed by mass spectrometry and 1H NMR. 32P post-labeling and co-chromatography with the DNA-styrene-7,8-oxide reaction products resulted in the assignment of adduct number 4 as O6-(2-hydroxy-2-phenylethyl)-2'--deoxyguanosine-3',5'-bisphosphate and adduct number 5 as O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3',5'-bisphosphate.

Topics: Chemical Phenomena; Chemistry; Deoxyguanine Nucleotides; DNA; Epoxy Compounds; Ethers, Cyclic; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phosphorus Radioisotopes

The 32P-postlabelling procedure has been used to detect styrene oxide (SO)-DNA adducts. Reactions of SO with DNA and dGMP in vitro produced adducts that were similar, indicating that dGMP was the primary base for modification in DNA. Two SO adducts were also detected in DNA isolated from lymphocytes of a styrene-exposed worker but not in DNA from an unexposed worker. These results indicate that 32P-postlabelling can be used for quantification of DNA adducts in workers exposed to styrene.

Topics: Deoxyguanine Nucleotides; DNA; Environmental Exposure; Environmental Monitoring; Epoxy Compounds; Ethers, Cyclic; Humans; Lymphocytes; Phosphorus Radioisotopes

In vitro reaction of DNA with styrene-7,8-oxide (styrene oxide) produced five adducts, as determined by 32P-postlabeling. When styrene oxide was reacted in vitro with deoxyribonucleotides, five adducts were observed from 2'-deoxyguanosine-3'-monophosphate, two from 2'-deoxyadenosine-3'-monophosphate, none from 2'-deoxythymidine-3-monophosphate or 2'-deoxycytidine-3'-monophosphate. Chromatographic comparison of the adducts formed in DNA with those formed with the deoxyribonucleotides suggests that deoxyguanosine is the primary site of DNA modification. Treatment of 9L cells with 1 mM styrene oxide resulted in the formation of several DNA adducts as detected by the postlabeling procedure. Our results indicate that 32P-postlabeling can be used to investigate DNA adducts formed by styrene oxide.

Topics: DNA; Epoxy Compounds; Ethers, Cyclic; Guanine; Phosphorus Radioisotopes