Chlorophyllin Chemoprevention in Trout Initiated by Aflatoxin B1 Bath Treatment: An Evaluation of Reduced Bioavailability vs. Target Organ Protective Mechanisms

Abstract

Chlorophyllin (CHL) is known to inhibit DNA adduction and hepatocarcinogenesis in trout when administered at doses up to 4000 ppm in the diet with aflatoxin B1 (AFB1). The principal protective mechanism is believed to involve CHL:AFB1 complex formation, which may reduce systemic carcinogen absorption. However, mechanisms operative within the target organ in situ have not been ruled out. The present study used alternative CHL and AFB1 exposures as well as hepatic metabolism studies to distinguish these mechanisms. Duplicate lots of 150 rainbow trout each were initiated by brief water bath exposure to 0.1 ppm AFB1, with or without 500 ppm CHL in the water. The addition of 500 ppm CHL to the water bath, under conditions where AFB1 is calculated to be >99% sequestered as the CHL:AFB1 complex, reduced hepatic AFB1-DNA adduction by 95% and reduced hepatocarcinogenesis from 20.5% to 2%, compared with exposure to AFB1 alone. Inclusion of 500 ppm CHL in the water bath also significantly reduced total body burden and hepatic levels of AFB1 as well as AFB2, a structural analogue of AFB1 unable to directly form the 8,9-epoxide proximate electrophile but equally capable of complexing with CHL. By contrast, internal target organ CHL loading by pretreatment of trout with 4000 ppm dietary CHL for 7 days prior to (and 2 days following) AFB1 waterbath exposure had no effect on AFB1-DNA adduction or tumorigenicity. Dietary CHL up to 8000 ppm had no effect on hepatic CYP2K1, CYP1A, glutathione transferase, UDP-glucuronosyl transferase, or, with one exception, the relative ratios among hepatic AFB1 metabolites in vivo. These results support the hypothesis that CHL:AFB1 complex formation and reduced systemic AFB1 bioavailability is a principal mechanism for CHL chemoprevention in this model and that in situ target organ inhibitory mechanisms are relatively insignificant.