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

Chlorophyllin (CHL), a food-grade derivative of the green plant pigmen t chlorophyll, has recently been shown in this laboratory to be a pote nt inhibitor in vivo of hepatic aflatoxin B-1 (AFB(1))-DNA adduction a nd 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 porphyrin s chlorine e6, protoporphyrin IX, and zinc protoporphyrin IX were also of the same order of magnitude as that of the commercial CHL. Mole ra tio 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 as sociation energy of -20 kcal/mol, independent of oxidation state of th e 8,9-double bond of AFB(1). AFB(1) incubated in vitro with liver micr osomes in the presence of added CHL showed comparable levels of inhibi tion in the production of several phase 1 metabolites, including the p ostulated procarcinogenic metabolite AFB 1 8,9-epoxide. Kinetic analys is of microsome-catalyzed AFB(1)-DNA adduction suggested a CHL inhibit ion constant near 10 mu M chlorin. In vivo, addition of CHL to concent rated AFB(1) solutions followed by gavage administration resulted in d ose-dependent inhibition of hepatic AFB(1)-DNA adduction, whereas the same dosages of AFB(1) and CHL incorporated into a single bolus of tro ut diet for gavage provided less protection at ah CHL doses. This obse rvation demonstrates that prior or prolonged CHL treatment is not requ ired for its antigenotoxic activity in vivo, but suggests that the eff iciency of CHL protection may depend on sample formulation. These find ings support a role for CHL-AFB(1) complex formation in CHL anticarcin ogenesis. Since the CHL precursor chlorophyll is present at very high concentration in certain green vegetables such as spinach, complex for mation between CHL-like compounds and carcinogens having at least part ially planar aromatic structure may contribute to the chemopreventive activities associated with a high green vegetable intake.