DETERMINING N7-ALKYLGUANINE ADDUCTS BY IMMUNOCHEMICAL METHODS AND HPLC WITH ELECTROCHEMICAL DETECTION - APPLICATIONS IN ANIMAL STUDIES AND IN MONITORING HUMAN EXPOSURE TO ALKYLATING-AGENTS
Jhm. Vandelft et al., DETERMINING N7-ALKYLGUANINE ADDUCTS BY IMMUNOCHEMICAL METHODS AND HPLC WITH ELECTROCHEMICAL DETECTION - APPLICATIONS IN ANIMAL STUDIES AND IN MONITORING HUMAN EXPOSURE TO ALKYLATING-AGENTS, Environmental health perspectives, 99, 1993, pp. 25-32
Many xenobiotics exert their toxic effects through interaction with DN
A in the cells of the exposed organism. This interaction may lead to t
he formation DNA adducts. Some of these may give rise to mutations tha
t initiate cell transformation and, ultimately, the formation of tumor
s. Sensitive methods for determining DNA adducts are indispensable for
the study of chemical mutagenesis and carcinogenesis and for biomonit
oring human exposure to genotoxic agents. Alkylating agents form an im
portant class of genotoxic compounds. They react preferentially at the
N7-position of guanine. Under neutral or acidic conditions, the adduc
ts can be readily released from the DNA backbone as the free base N7-a
lkylguanine (N7-AlkGua). The imidazole ring of N7-alkyldeoxyguanosine
(N7-AlkdGuo) can be opened under alkaline conditions, which results in
formation of a more stable adduct in DNA. To develop immunochemical m
ethods for the detection of N7-alkylations, we immunized mice with var
ious alkylguanosines in the ring-opened form (RON7-AlkdGuo). Antibodie
s were selected to detect adducts in isolated DNA by competitive ELISA
and in single cells by immunofluorescence microscopy (IFM). Various m
onoclonal antibodies were characterized in detail with respect to spec
ificity and sensitivity toward methylated, ethylated, and hydroxyethyl
ated DNAs. The antibodies showed extensive cross-reactivity toward N7-
(m)ethyl- and N7-(2-hydroxyethyl)guanine modifications in the ring-ope
ned form. The limits of detection in the direct and competitive ELISA
were 5-10 and 1-2 adducts per 10(6) nucleotides, respectively. The det
ection limit of the IFM method was about 20 adducts per 10(6) nucleoti
des. To calibrate the immunochemical methods, we used an HPLC with ele
ctrochemical detection (HPLC-EC) to quantify N7-methyl-, N7-ethyl-, an
d N7-(2-hydroxyethyl)guanine released from DNA by neutral thermal hydr
olysis. The base adducts were separated from nonmodified guanine and a
denine on a reverse-phase HPLC column and quantified by use of an EC-d
etector at an oxidation potential of 1.35 V. For each of the alkylated
guanines mentioned, the detection limit was about 0.3 adducts per 10(
6) nucleotides. Results are presented of the application of the immuno
chemical assays and HPLC-EC to investigate the induction of methylatio
ns in liver DNA of rats treated in vivo with low dosages of hydrazine
and the induction of DNA methylations in nucleated peripheral blood ce
lls collected from a patient who received chemotherapy with the methyl
ating cytostatic drug dacarbazine.