Direct synthesis of aflatoxin B₁‐N⁷ guanine adduct: A reference standard for biological monitoring of dietary aflatoxin exposure in molecular epidemiological studies

Abstract

Aflatoxin B₁‐N⁷‐guanine and aflatoxin B₁‐human serum albumin adducts have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synthesize aflatoxin B₁‐8,9‐epoxide in vitro and its subsequent interaction with DNA or synthetic oligodeoxynucleotide was used as a source of authentic aflatoxin B₁‐N⁷‐guanine adduct. In the present communication we report a simple single step procedure for the synthesis of aflatoxin B₁‐N⁷‐guanine adduct using free guanine and m‐chloroperbenzoic acid as the chemical oxidant for the production of AFB₁‐8,9‐epoxide. At a molar ratio of 1:1 of AFB₁‐8,9‐epoxide and guanine the recovery of the AFB₁‐N⁷‐guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB₁‐N⁷‐guanine adduct was found to be low (30–40%). HPLC analysis of the AFB₁‐N⁷ guanine adduct showed a retention time identical with the retention time of the AFB₁‐N⁷‐guanine adduct synthesized using calf thymus DNA. TLC‐fluorodensitometric analysis indicated that the R _f of the AFB₁ ‐N ⁷‐guanine adduct was zero. Spectral analysis of the adduct synthesized showed an excitation wavelength of 360 nm and emission wavelength at 440 nm in phosphate buffer (100 mM, pH 7.4). Further, the formation of the AFB₁‐N ⁷‐guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB₁‐N ⁷‐guanine adduct thus synthesized was stable in both acidic as well as lyophilized conditions over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA‐guanine‐N ⁷‐AFB₁ also cross‐reacted with calf thymus DNA‐AFB₁ adduct, indicating specificity to the guanine‐N ⁷‐AFB₁ moiety. The method developed may find immediate application as a source of authentic reference standard in molecular epidemiological studies.

Keywords:

• aflatoxin B₁
• guanine
• aflatoxin B₁‐N⁷‐guanine adduct
• molecular biomarker
• epidemiology

Notes

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