Mechanisms of Chlorophyllin Anticarcinogenesis: Dose–Responsive Inhibition of Aflatoxin Uptake and Biodistribution Following Oral Co-administration in Rainbow Trout

Abstract

Chlorophyllin (CHL) is a potent blocking agent against aflatoxin B1 DNA adduction and tumorigenesis in the trout model, but mechanisms responsible for this chemoprotection in vivo are not well established. This study employed aflatoxin B2 (AFB2), a structural analogue of AFB1 that cannot be metabolized directly to the 8,9-exo-epoxide electrophile, to investigate CHL effects on carcinogen uptake and distribution kinetics following oral exposure in trout. CHL was shown to form an AFB2 complex in vitro with a dissociation constant (Kd = 1.92 ± 0.13 μM) comparable to that with AFB1. Following gavage, [3H]AFB2 equivalents distributed rapidly from the stomach to other organs including blood, liver, and eventually to bile as a major repository. Bile was found to contain almost entirely parent AFB2 1 h after gavage, with a single metabolite dominating 3–24 h and an additional metabolite prominent by 48 h after gavage. Addition of sufficient CHL (≥13.9 mM) to assure >99% complexation of AFB2 (0.906 μM) in the gavage mix resulted in 80–90% reduction in AFB2 equivalents in liver and bile 3 h after gavage. In three separate kinetic studies of up to 120 h postgavage, addition of ≥13.9 mM CHL to the gavage mix reproducibly and markedly delayed the rate of AFB2 loss from stomach, retarded its appearance in blood, liver, and bile, and reduced peak AFB2 concentrations in those tissues by up to 60%. Introduction of a food bolus immediately after gavage prolonged AFB2 residence in stomach and intestine but did not abrogate the inhibitory effects of CHL on AFB2 uptake and distribution. These results demonstrate that oral co-treatment with CHL under conditions where complex formation is initially assured, substantially reduces AFB2 systemic uptake and target organ bioavailability in the trout.