DIRECT SYNTHESIS OF AFLATOXIN B-1-N-7 GUANINE ADDUCT - A REFERENCE-STANDARD FOR BIOLOGICAL MONITORING OF DIETARY AFLATOXIN EXPOSURE IN MOLECULAR EPIDEMIOLOGIC STUDIES

Aflatoxin B-1-N-7-guanine and aflatoxin B-1-human serum albumin adduct s have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synth esize aflatoxin B-1-8,9-epoxide in vitro and its subsequent interactio n with DNA or synthetic oligodeoxynucleotide was used as a source of a uthentic aflatoxin B-1-N-7-guanine adduct. In the present communicatio n we report a simple single step procedure for the synthesis of aflato xin B-1-N-7-guanine adduct using free guanine and m-chloroperbenzoic a cid as the chemical oxidant for the production of AFB(1)-8,9-epoxide. At a molar ratio of 1:1 of AFB(1)-8,9-expoxide and guanine the recover y of the AFB(1)-N-7-guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB(1)-N-7- guanine adduct was found to be low (30-40%). HPLC analysis of the AFB( 1)-N-7 guanine adduct showed a retention time identical with the reten tion time of the AFB(1)-N-7-guanine adduct synthesized using calf thym us DNA. TLC-fluorodensitometric using calf thymus DNA. TLC-fluorodensi tometric analysis indicated that the R-f of the AFB(1)-N-7-guanine add uct was zero. Spectral analysis of the adduct synthesized showed an ex citation wavelength of 360 nm and emission wavelength at 440 nm in pho sphate buffer (100 nM, pH 7.4). Further, the formation of the AFB(1)-N -7-guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB(1)-N-7-guanine adduct thus synthesized was stable in both acidic as well as lyophilized condition s over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA-guanine-N-7-AFB(1) also c ross-reacted with calf thymus DNA-AFB(1) adduct, indicating specificit y to the guanine-N-7-AFB(1) moiety. The method developed may find imme diate application as a source of authentic reference standard in molec ular epidemiological studies.