Inhibition of dibenzo[a,l]pyrene-induced multi-organ carcinogenesis by dietary chlorophyllin in rainbow trout

Abstract

Cancer chemoprevention by dietary chlorophyllin (CHL) was investigated in a rainbow trout multi-organ tumor model. In study 1, duplicate groups of 130 juvenile trout were treated for 2 weeks with control diet, 500 p.p.m. dibenzo[a,l]pyrene (DB[a,l]P) or 500 p.p.m. DB[a,l]P + 2052 p.p.m. CHL, then returned to control diet. DB[a,l]P alone proved somewhat toxic but induced high tumor incidences in liver (61%), stomach (91%) and swimbladder (53%) 11 months after initiation. CHL co-feeding abrogated DB[a,l]P acute toxicity and reduced tumor incidences to 18% in liver, 34% in stomach and 3% in swimbladder (P </= 0.01). A second tumor and DNA adduct study using a non-toxic initiation protocol (200 p.p.m. DB[a,l]P +/- 4000 p.p.m. CHL for 4 weeks) confirmed these results. Potential CHL inhibitory mechanisms were investigated. Dietary CHL inhibited hepatic DB[a, l]P-DNA adducts in the two tumor studies by 89 and 76%, respectively. CHL was shown to complex strongly with DB[a,l]P (K(d1,2) = 1.59 +/- 0.01 microM, stoichiometry 2CHL:DB[a,l]P) and strongly inhibited DB[a,l]P mutagenesis in the Salmonella assay. Significant inhibition occurred at CHL concentrations substantially less than stoichiometric with DB[a,l]P and thus not reflecting simple DB[a,l]P sequestration via complexation. These initial findings suggest that CHL chemoprevention reflects complexation that might limit DB[a,l]P uptake in vivo, antimutagenic mechanisms such as catalytic degradation of the proximate electrophile in target cells, or both. These results demonstrate that dietary CHL is a reproducibly effective chemopreventive agent for DB[a,l]P multi-organ tumorigenesis in trout and suggest that reduced DB[a,l]P-DNA adducts may be predictive biomarkers of CHL reduction of DB[a,l]P-initiated hepatic tumors.

Fig. 1

Summary of the antimutagenic and anticarcinogenic effects of CHL in trout. (a) and (b) were modified from the original results in Ref. [4] and elsewhere (R.H. Dashwood, unpublished data), whereas (c) was obtained by re-plotting of the original findings in Ref. [2].

Fig. 2

Spectrophotometric titration of AFB₁ with CHL. Starting substrate (AFB₁)concentration, 20 μM in 0.1 M sodium phosphate buffer, pH 7.4; 25°C; ligand concentration, 7.5 μM CHL (each addition); b = 1 cm. From the Benesi–Hildebrand plot, K_d = 1.92 μM (see Ref. [8] for details of the methodology).

Fig. 3

Computer generated molecular model of the AFB₁-CHL complex. Energy-minimized molecular models of the interaction between AFB₁ and CHL were obtained using HyperChem, as described previously for the complexes between chlorophylls and heterocyclic amines [14] or polycyclic aromatic hydrocarbons [11]. (a) Top view showing the overlapping ring systems of AFB₁ (yellow) and CHL; (b) side view showing the 8,9-double bond of AFB₁ (upper left) oriented 180° with respect to the carboxyl groups in CHL (right); minimized energy of the complex shown, −19.023 kcal/mol.

Fig. 4

Microinjection of chlorophyll a into an egg containing rainbow trout embryo 21 days post-fertilization. The egg was injected with 1 μl of vehicle (ethanol:DMSO, 1:1, v/v) containing 1000 μg chlorophyll a.