Detection and characterization of two major ethylated deoxyguanosine adducts by high performance liquid chromatography, electrospray mass spectrometry, and 32P-postlabeling. Development of an approach for detection of phosphotriesters.

Postlabeling can be one of the most sensitive methods for the measurement of DNA adducts. However, for the determination of alkylated adducts, essential requirements are standards which must be fully chemically characterized. In order to develop a postlabeling assay for monitoring exposure to genotoxic ethylating agents, the reaction of diethyl sulfate with 2'-deoxynucleoside 3'- and 5'-monophosphates was examined. The adducts generated were fully characterized using HPLC, electrospray tandem mass spectrometry, UV, and postlabeling. The major product was the phosphodiester derived from alkylation of the phosphate, and alkylation of the base occurred to a lesser extent. The phosphodiester standard, 2'-deoxyguanosine 3'-(mono-O-ethyl phosphate) (3'Et-pdG), was used to develop a postlabeling assay for the detection of this adduct in DNA samples. Since alkylated phosphodiesters in DNA are not susceptible to the actions of micrococcal nuclease and calf spleen phosphodiesterase, they can be obtained as alkylated phosphodiester dinucleosides from DNA. Nuclease P1 was used as enhancement step which allowed the separation of these adducted phosphotriesters from the unmodified nucleotides by HPLC. Subsequent hydrolysis of the phosphotriester dinucleosides in alkali yielded phosphodiesters, including 3'Et-pdG, which was efficiently postlabeled. This approach was shown to be capable of detecting this adduct in liver DNA from mice treated intraperitoneally with N-nitrosodiethylamine.

Mutation and formation of methyl- and hydroxylguanine adducts in DNA caused by N-nitrosodimethylamine and N-nitrosodiethylamine with UVA irradiation.

Previously we reported that when Escherichia coli was treated with N-nitrosodialkylamine under irradiation with near UV light, mutagenesis of the bacteria took place: there was no requirement for metabolic activation. We have now studied the spectra of mutations caused by N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) with UVA (320-400 nm) irradiation, using standard tester strains for identifying types of mutations. Induced mutations by NDMA + UVA were the transition GC-->AT and transversions GC-->CG, GC-->TA and AT-->TA. NDEA + UVA induced mainly the GC-->CG transversion. In both cases no frameshift mutations were observed. When O6-alkylguanine-DNA alkyltransferase-deficient strains of E. coli and Salmonella typhimurium were used, the mutation levels with both NDMA + UVA and NDEA + UVA became remarkably higher than those observed with the proficient strains. We measured the O6-methylguanine (O6-meG) level in calf thymus DNA treated with NDMA + UVA. The O6-meG level was increased as a function of NDMA concentration and irradiation time. We also detected N7-methylguanine in DNA treated with NDMA + UVA. In our previous work we found formation of 8-oxodeoxyguanosine (8-oxodG) in DNA treated with N-nitrosomorpholine + UVA. The 8-oxodG/dG ratio in DNA treated with NDMA + UVA increased up to 42-fold over that of the untreated control and that in DNA treated with NDEA + UVA increased up to 67-fold. 8-OxodG formation was not affected by replacing H2O in the reaction mixture with D2O, suggesting that singlet oxygen is not the rate limiting factor in this photoactivation. We conclude that both alkylation and oxidation are involved in mutations induced by NDMA + UVA and NDEA + UVA.

Monitoring of exposure to acrylonitrile by determination of N-(2-cyanoethyl)valine at the N-terminal position of haemoglobin.

A selected ion monitoring gas chromatography-mass spectrometry (GC-MS) procedure was developed to determine the interaction product formed by acrylonitrile (ACN) with the N-terminal amino group in haemoglobin. The product, N-(2-cyanoethyl)valine (CEV), was analysed following its release from the protein by a modified Edman degradation procedure. Quantitation was achieved using N-(2-cyanoethyl)-[2H8]Val-Leu-Ser as internal standard. The limit of detection of the assay was 1 pmol CEV/g globin. A close to linear dose-response relationship was found for adduct formation in rats treated with ACN by gavage. On the basis of a linear extrapolation, a dose of 1 mg/kg body wt yielded 248 pmol CEV/g globin. Two groups of workers who were exposed to ACN contained 1984 +/- SD 2066 (n = 9) and 2276 +/- SD 1338 (n = 7) pmol CEV/g globin respectively. These values were highly significantly greater (P < 0.01 following a one-way analysis of variance with a logarithmic transformation of the data) than those in a group of control workers in the same factory (31.1 +/- SD 18.5 pmol CEV/g globin, n = 11). The concentrations of N-terminal CEV in globin samples from 13 smoking and 10 non-smoking mothers and from their newborns were determined. Adduct levels in the smokers averaged 217 +/- 85.1 pmol CEV/g globin, significantly higher than the levels in non-smokers, which were undetectable. Individual values in the mothers were very highly correlated with the levels in their babies (which averaged 99.5 +/- 53.8 pmol CEV/g globin), which demonstrates that transplacental transfer of ACN occurs. Significant correlations were also found between the number of cigarettes smoked per day by the mother and the CEV levels in both the mothers' and newborns' globin. There was, however, no correlation between the CEV levels and those of the ethylene oxide adduct N-(2-hydroxyethyl)valine in samples from either the mothers or babies.

Synthesis and characterization of peptide adducts for use in monitoring human exposure to acrylonitrile and ethylene oxide.

Human exposure to ethylene oxide and acrylonitrile may be monitored by determination of the products that these electrophilic compounds form with the amino terminal of hemoglobin. The procedure involves a modified Edman degradation, using the reagent pentafluorophenyl isothiocyanate, which cleaves the adducted N-terminal amino acid from the protein chain as a substituted pentafluorophenyl thiohydantoin. This may then be quantitated using selected ion monitoring gas chromatography-mass spectrometry (GC-MS). To overcome problems arising from the lack of an ideal internal standard for this analysis, we have now synthesized adducts of ethylene oxide and acrylonitrile with the N-terminal tripeptide of the alpha-chain of human globin (Val-Leu-Ser). These adducts should behave analogously to the protein adduct in the Edman degradation. The adducts were characterized by nuclear magnetic resonance and fast atom bombardment and electrospray MS and tandem MS. Additionally, an analogous stable isotope-labeled standard was synthesized for the acrylonitrile adduct using a d8-labeled tripeptide. Quantitative calibration lines for analysis of the adducts have been established and the validity of the assays established by the detection of acrylonitrile adducts in the globin of acrylonitrile-exposed workers and of cigarette smokers.

Sequencing of oligonucleotides using high performance liquid chromatography and electrospray mass spectrometry.

We report the sequence analysis of an oligodeoxyribonucleotide by electrospray mass spectrometry (ES-MS). For full sequence analysis of the oligomer, d(CGTAGCTACG), an enzyme digestion step followed by off-line high performance liquid chromatography was used. For ES-MS analysis, introducing the sample in an imidazole buffer with 70% acetonitrile increased the electrospray ion currents and helped reduce sodium-adduct ion peaks.

Electrospray mass spectrometry of Malayan pit viper (Calloselasma rhodostoma) venom.

A high-performance liquid chromatography protocol has been developed for the analysis of snake venoms. This system has been used to isolate eight fractions from Malayan pit viper (Calloselasma rhodostoma) venom. The fractions have been analysed using electrospray mass spectrometry. A number of major components were found with masses ranging from 13,670 to 22,750 Da.