Method development and analysis of O(6) methylguanine adducts in human peripheral lymphocytes.
DeBord-DG; Werren-DM; Cheever-KL; Reid-TM; Butler-MA; Walker-CV; Reh-BD
Toxicologist 1998 Mar; 42(1-S):181
A method was developed to measure O(6)-methhylguanine adducts in DNA from human peripheral lymphocytes obtained from workers exposed to nitrosamines, such as nitrosodimethylamine. Lymphocytes were isolated from whole blood samples using Histopaque(R). After isolation, DNA was enzymatically hydrolyzed to nucleotides. O(6)-Methyldeoxyguanosine (O6mdG) was separated from its RNA counterpart, 06-methylguanosine, by HPLC using a Beckman Altex Ultrasphere ion pair column and UV detection at 254 nm with the mobile phase being O.IM triethylamine acetate using a gradient of I to 10% acetonitrile. O6mdG eluted between 40-45 minutes, while O6- methylguanosine eluted after 48 minutes. The 06mdG fraction was concentrated and hydrolyzed to O6-methylguanine (O6mG) in O.1N HCI for 1 hour at 60 degrees C. The deoxyguanosine (dG) fraction was also collected - reference standard, concentrated, but was not acid hydrolyzed. After concentration the levels of O6mG and dG were determined using HPLC with electrochemical detection using a C-I8 reverse-phase column with 100mM sodium acetate and 5% methanol, pH 5.2 as a mobile phase at lml/min. Electrochemical conditions for detection varied with 06mG run at 650 mY, while dG was run at 800mV 06mG ranged from 0.03 to 9.8 06mG adducts/10(7) dG nucleotides. 06meG could not be detected in every worker and only five controls had measurable levels of 06meG. Consequently, there was a relationship in O6meG adduct levels in workers based on exposure categories defined in NIOSH HETA 9'-0072. N(7)-methyldeoxyguanosine levels were measured in the same samples by the 32 p postlabeling assay. Although somewhat less sensitive than the (32p) postlabeling assay, the electrchemical method is less labor intensive and lends itself to automation for analysis of a large numbers of samples. In addition this method allows for separation and quantitation of a number of alkylated adducts from the same sample.
DNA-damage; Nitrosamines; Lymphocytes; Ribonucleic-acids; Electrochemical-analysis; Deoxyribonucleic-acids
The Toxicologist. Society of Toxicology 37th Annual Meeting, March 1-5,1998, Seattle, Washington