MECHANISMS OF CHLOROPHYLLIN ANTICARCINOGENESIS AGAINST AFLATOXIN B-1 - COMPLEX-FORMATION WITH THE CARCINOGEN

Chlorophyllin (CHL), a food-grade derivative of the green plant pigment chlorophyll, has recently been shown in this laboratory to be a potent inhibitor in vivo of hepatic aflatoxin B-1 (AFB(1))-DNA adduction and hepatocarcinogenesis (Breinholt et al. (1995) Cancer Res. 55, 57-62). We report here that CHL forms a strong noncovalent complex with AFB(1) in vitro (dissociation constant (K-d) by Scatchard analysis = 1.4 (+/- 0.4) mu M based on copper chlorin content), which may contribute to its anticarcinogenic activity. K-d values for the related porphyrins chlorine e6, protoporphyrin IX, and zinc protoporphyrin IX were also of the same order of magnitude as that of the commercial CHL. Mole ratio analysis provided evidence that all porphyrins examined associate with AFB(1) at an approximate one to one stoichiometric ratio. Energy minimization computer modeling of the complex indicates a favorable association energy of -20 kcal/mol, independent of oxidation state of the 8,9-double bond of AFB(1). AFB(1) incubated in vitro with liver microsomes in the presence of added CHL showed comparable levels of inhibition in the production of several phase 1 metabolites, including the postulated procarcinogenic metabolite AFB 1 8,9-epoxide. Kinetic analysis of microsome-catalyzed AFB(1)-DNA adduction suggested a CHL inhibition constant near 10 mu M chlorin. In vivo, addition of CHL to concentrated AFB(1) solutions followed by gavage administration resulted in dose-dependent inhibition of hepatic AFB(1)-DNA adduction, whereas the same dosages of AFB(1) and CHL incorporated into a single bolus of trout diet for gavage provided less protection at ah CHL doses. This observation demonstrates that prior or prolonged CHL treatment is not required for its antigenotoxic activity in vivo, but suggests that the efficiency of CHL protection may depend on sample formulation. These findings support a role for CHL-AFB(1) complex formation in CHL anticarcinogenesis. Since the CHL precursor chlorophyll is present at very high concentration in certain green vegetables such as spinach, complex formation between CHL-like compounds and carcinogens having at least partially planar aromatic structure may contribute to the chemopreventive activities associated with a high green vegetable intake.