Aflatoxin B₁ (AFB₁) is known to exert species-dependent toxicity and carcinogenicity that may be related to its metabolism and formation of DNA adducts in the respective species.The first part the study focused on the kinetics of AFB₁ metabolism in primary hepatocytes of mouse, rat and human origin by HPLC-MS/MS analysis. Selective and sensitive quantification of AFB1 and its metabolites such as aflatoxin P₁ (AFP₁), aflatoxin M₁ (AFM₁), aflatoxin Q₁ (AFQ₁), aflatoxicol (AFL), AFB₁-N-acetylcysteine, AFB₁-glutathione (AFB₁-GSH), AFB₁-guanine and AFB₁-lysine in cells and cell medium. Upon treatment of cells with AFB₁, samples were collected up to 24 h. In addition, the cellular DNA was analyzed for adducts (AFB₁-guanine) which are linked to the carcinogenicity and mutagenicity of AFB₁.
Fast metabolization and comparably high levels of the far less toxic AFP₁ as main metabolite were observed for mice hepatocytes. Furthermore, AFB₁-GSH and AFM₁ were formed quickly, and only low amounts of AFB₁-DNA adducts were detected, which disappeared almost completely from the DNA up to 24 h. In contrast, rat hepatocytes metabolized AFB₁ significantly slower and formed mainly AFM₁ and AFB₁-GSH. Compared to mouse hepatocytes higher amounts of AFB₁-DNA adducts were detected, explaining the higher carcinogenic potency of AFB₁ in rats in comparison to mice. The main human metabolites were AFM₁ as well as AFQ₁ and AFL, which had only a minor role in the other species.
In the second part AFB₁ was applied to mice and rats, and samples from plasma, urine and bile were collected for up to 24 h. The metabolite pattern of this in vivo time-course showed comparable results to the in vitro experiments with primary rodent hepatocytes. In order to analyse also the tissue distribution a two-photon microscopy-based technique for intravital imaging of AFB₁ based on its blue fluorescence was applied. The results show a very rapid uptake of AFB₁ in hepatocytes of mice and enrichment in the nuclei with kinetic differences between mice and rats.
In summary, the results demonstrate a strong correlation between the sensitivity of certain species towards AFB₁ and their respective metabolism and distribution.

Hans-Ulrich Humpf

Univ Muenster

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