Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout

Abstract

We recently reported that chlorophyll (Chl) strongly inhibits aflatoxin B(1) preneoplasia biomarkers in rats when administered by co-gavage (Simonich et al., 2007. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Carcinogenesis 28, 1294-1302.). The present study extends this by examining the effects of dietary Chl on tumor development, using rainbow trout to explore ubiquity of mechanism. Duplicate groups of 140 trout were fed diet containing 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1000-6000 ppm Chl, for 4 weeks. DBP induced high tumor incidences in liver (51%) and stomach (56%), whereas Chl co-fed at 2000, 4000 or 6000 ppm reduced incidences in stomach (to 29%, 23% and 19%, resp., P<0.005) and liver (to 21%, 28% and 26%, resp., P<0.0005). Chlorophyllin (CHL) at 2000 ppm gave similar protection. Chl complexed with DBP in vitro (2Chl:DBP, K(d1)=4.44+/-0.46 microM, K(d2)=3.30+/-0.18 microM), as did CHL (K(d1)=1.38+/-0.32 microM, K(d2)=1.17+/-0.05 microM), possibly explaining their ability to inhibit DBP uptake into the liver by 61-63% (P<0.001). This is the first demonstration that dietary Chl can reduce tumorigenesis in any whole animal model, and that it may do so by a simple, species-independent mechanism.

Fig. 1

Spectrofluorometric titration of DBP with chlorophyll (Chl). (A) Effect of Chl on the DBP emission spectrum from 400 to 500 (± 8) nm (excitation 318 ± 8 nm) with DBP (substrate) concentration at 1.18μ. Chl (ligand) was added in 0.4 μM increments up to 10 μM (some titrations omitted from the figure for clarity) and the spectrum was recorded 2 minutes after each addition. (B) Quantification of Chl quenching of DBP fluorescence at 425 ± 8 nm recorded from the above spectra. Data were normalized by converting fluorescence units to ΔF/F₀ and the data was fitted to the 2Chl:DBP complexation model.

Fig. 2

Pharmacokinetics of 200 μM [¹⁴C]-DBP following oral gavage treatment of 0.02 μCi/g body weight. Chemoprotective treatments included 2 mM chlorophyllin or 2 mM chlorophyll. Ten fish were killed at each time point after gavage Samples of each tissue were individually collected, processed and evaluated by liquid scintillation counting for ¹⁴C activity. Data from the pyloric cecae and the lower intestine were combined into one compartment termed the intestinal tract. ● = DBP, ○ = DBP + chlorophyllin, ▾ = DBP + chlorophyll. Each data point represents mean ± SE of the 10 samples.

Fig. 3

Low-temperature ESR spectra of chlorophyllin with or without ascorbate and/or bathophenanthroline disulphonate (BCS). ESR measurements were carried out in 0.1 M phosphate buffer, pH 7.4, at 77K as described in Materials and Methods. The reaction mixtures contained 0.5 mM chlorophyllin, 2 mM ascorbate, or 2 mM ascorbate plus 10 mM BCS.